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1. The dialog will appear Find Qualifier Search in CDS Qualifier Name Value Match Parameter Exact match Contains substring Here you can specify the name and the value of the qualifier and select the searching parameter Exact match or Contains substring Deleting Annotations and Qualifiers Selected annotations groups and qualifiers can be deleted using the Delete key To remove an annotation object from the active view select the object in the Annotations editor and press the Shift Delete Note that the object will not be removed from the project but just from the active Sequence View To add object again just drag and drop it to the Sequenc e View Importing Annotations from CSV It is possible to import annotations for a sequence from an annotations table stored in the CSV format To import annotations from a CSV file right click on a Project View and select mport Import annotations from CSV The following dialog box will appear 19 Unipro UGENE Manual Version 1 20 0 m Import Annotations from CSV File to read Results Result file emas Add result file to project Column separator value hex 2c length 1 File parsing Column separator Script Edit First lines to skip Do not skip Skip all lines starts with the text Interpret multiple separators like a single separator try when separator is a whitespace character Remove quotes Default annotation name
2. To search data in the nucleotide or protein databases enter a general text query to the search field select the database and click on the Sear ch button You can use a protein name gene name or gene symbol directly Searching with a submitter or author name in the following format will produce the best results Use the boolean operator AND to find records that contain every one of your search terms the intersection of search results Use the boolean operator OR to find records that include one of several search terms the union of search results Use the boolean operator NOT to exclude records matching a search term To limit results use the Result limit field After you click the Search button UGENE searches the biological objects and shows it in the Results field You can download the object s Select one or several objects for selecting several objects use the Ctr button and click the Download button The dialog will appear 36 Unipro UGENE Manual Version 1 20 0 m Fetch Data from Remote Database Resource ID Kl690776 Database Save to directory output oma After you click the OK button UGENE downloads the biological objects and adds it to the current project Fetching Data from Remote Database UGENE allows fetching data from remote biological databases such as NCBI GenBank NCBI protein sequence database and some others To fetch data select the File Access remote database item in the main menu The d
3. Building Dotplot for Currently Opened Sequence Navigating in Dotplot To zoom in zoom out a dotplot you can e Rotate the mouse wheel e Press corresponding zoom buttons located on the left To move the zoomed region you can aauanbes 82610 ON 65k Og SPE Ss Mihimap ae o NC_014267 sequence min length 11 identity 100 FOk FAK e Hold the middle mouse button and move the mouse cursor over the zoomed region of the doplot e Click on the desired region of the minimap in the right bottom corner e Activate the Scroll tool hold the left mouse button and move the mouse cursor over the zoomed region Zooming to Selected Region To select a dotplot region activate the Select tool hold down the left mouse button and drag the mouse cursor over the dotplot When you select a region on a dotplot the corresponding region is also selected in other Sequence View areas Sequence details view Sequ 110 Unipro UGENE Manual Version 1 20 0 ence zoom view etc The opposite is true as well if you select a region in a Sequence View area the corresponding region is also selected in the dotplot view To zoom to the region selected click the Zoom in on the left 12 230 16k 918k 20 _ _ _ Click it to zoom to 3 the selected region w 15k antl 10k Eril Selecting Repeat To select a repeat activate the Select tool and click on the repea
4. Name vale S Annotations result gb at 0 2 G ai 19 0 the_qualifier ppe Sma 10 20 i the_qualifier pp Exporting Annotations Open the Sequence View with document that contains annotations Select a single or several annotations or annotation groups in the Annota tion editor select the Export Export annotations context menu item The Export Annotations dialog will appear m Export Annotations Export to file C work ugene data samples Genbank Cvu55762_annotations csw File format csv Save sequences under annotations E Save Sequence names 78 Unipro UGENE Manual Version 1 20 0 Here you can set the path to the file choose the file format and optionally for CSV format you can save the sequence along with annotations and save sequence names 79 Unipro UGENE Manual Version 1 20 0 Sequence View Extensions The functionality of the Sequence View can be significantly increased with Sequence View Extensions Below is the demonstration its functionality The Circular Viewer shows the circular view of a sequence mRNA a a SOB59345 1 mRNA ee a mRNA repeat_region MRNA misc_binding s a a e misc_recomb kS k old_sequence 7 5 w Fa n T l mRNA ww AABSIS40 1 we exon 7SCPLASM sequence 6316 bp Tena misc_binding 7 conflict mRNA Tea exon MRENA S e in conflict eM MRNA ie Noh AAB59340 1 oy s repeat region E i h a ae 7 z ow Ka A es Conflict
5. Convert Alignment to Separate Sequences Export to file Fle format to use Add document to the project Gap characters Keep Here it is possible to specify the result file location to select a sequence file format to define whether to keep or remove gaps chars in the aligned sequences and optionally add the created document to the current project Exporting Nucleic Alignment to Amino Translation Select a single object with a nucleic sequence alignment in the Project View window and click the Export Export nucleic alignment to amino translation context menu item 30 Unipro UGENE Manual Version 1 20 0 File Actions Settings Tools Window Help Gores asg air eeGe TtAGttTacTAATTCGAGCtTG AA 6 a 10 i 14 16 16 20 ca LE 1 Project Open view Add to view Unload selected documents Lock document for editing Add Import Export Edit Export alignment to sequence format Export nucleic alignment to amino translation Remove 7 Export document og Save selected documents F p Lni 3 Coli 604 The Export Nucleic Alignmemt to Amino Translation dialog will appear Export Nucleic Alignment to Amino Translation Export to file C work ugene data samples CLUSTALW COL_transl alin re format to use Amino translation 1 The Standard Genetic Code Add document to the project Export range Whole alignment Selected rows Here it is possible to specify the result file
6. Criteria Valid Values 3 GC 50 60 Tm C 55 80 GC Clamp gt 1GorCat3 end Runs lt 4 base runs Self dimers Delta G 9 3 kcal mole Base Pairs 7 1 MII A Delta G 5 2 kcal mole Base Pairs 3 ARATE Hetero dimers Delta G 16 kcal mole Base Pairs 13 Tt Ut lt ttl This is a dialog with statistic details about primers melting temperature GC content dimers self dimers etc If a value is not correct for its criteria then it is colored in red Primer Library The primer library is a storage for keeping user primers The added primers are stored between UGENE sessions Go to the Too ls gt Primer gt Primer library context menu to configure the primer library The following window will appear 201 Unipro UGENE Manual Version 1 20 0 UGENE Primer Library File Actions Settings Tools Window Help content 45 Tm C Length bp Sequence Primer 1 52 63 GTCCCACTGTACGTTTACG tz L Project Test Primer 37 5 ACGTTTACGOTACGATTCGATACACATAGACAT No active tasks 9 Ez Name New Primer Input the primer sequence and primer name and click on the OK button Select the primer and click the Edit primer button to edit primer Select the primer in the table you can use Ctrl and Shift and click Remove primer s button to remove primer s To export primer s select it and click the Export primer s button The following dialog will appear Export Primers Export to Format
7. DNA sequence generator is a tool that generates a random DNA sequence with specified nucleotide content To generate a random DNA sequence select the Too ls gt Generate sequence item in the main menu The dialog will appear Window size Number of sequences E Initialize random generator manually Content Reference O van Output W Add to project The following parameters are available 171 Unipro UGENE Manual Version 1 20 0 Length length of the resulted sequence s using 1000 bp by default Window size size of window where set content using 1000 by default Number of sequences number of sequences to generate using 1 by default Initialize random generator manually value to initialize the random generator Reference path to the reference file could be a sequence or an alignment Manual set the base content persents To configure base content click on the Configure button and set base content manually Output file output file Format output file format using fasta by default Add to project adds the generated sequence s to project Once the Search button has been pressed the sequence s are created ORF Marker From this chapter you can learn how to search for Open Reading Frames ORF in a DNA sequence The ORFs found are stored as automatic annotations This means that if the automatic annotations highlighting has been enabled then ORFs are searched and highlighted fo
8. Mew annotation 3k 25k 3k 3 5k dk Copy Select E M Add ud Be Find pattern Ctrl F Align SW Find pattern Smith Waterman Ctrl Shift F 12 4 Clonning Find ORFs TITA Export I i Find annotated regions F Edit sequence TERRAS Build dotplot H Find repeats E Find tandems Name Find query designer pattern Find restriction sites Query NCBI BLAST database Search HMM signals with HMMERS Search with HMM model Search TFBS with SITECON Search TFBS with matrices Primers ae 6 DAO ORNS BMS Predict secondary structure Guerry wt AST Gery Mob BLASTA vwy lt iil For details see the next sections of the documentation e Circular Viewer e Circular View Settings e 3D Structure Viewer e Opening 3D Structure Viewer e Changing 3D Structure Appearance e Selecting Render Style e Selecting Coloring Scheme Calculating Molecular Surface Selecting Background Color Selecting Detail Level e Enabling Anaglyph View Moving Zooming and Spinning 3D Structure Selecting Sequence Region Selecting Models to Display Structural Alignment Exporting 3D Structure Image Working with Several 3D Structures Views e Chromatogram Viewer e Exporting Chromatogram Data e Viewing Two Chromatograms Simultaneously e DNA RNA Graphs Package e Description of Graphs e Graph Settings e Saving Graph Cuttoffs as Annotations e Doiplot e Creating Dotplo
9. This chapter gives an overview of the Alignment Editor components and explains basic concepts of browsing an alignment Alignment Editor Features Alignment Editor Components Navigation Coloring Schemes e Creating Custom Color Scheme Highlighting Alignment Zooming and Fonts Searching for Pattern Consensus e Export Consensus e Alignment Overview Alignment Editor Features The Alignment Editor is a powerful tool for visualization and editing DNA RNA or protein multiple sequence alignments The editor supports different multiple sequence alignment MSA formats such as ClustalW MSF and Stockholm The full list of file formats supported in UGENE is here The editor provides interactive visual representation which includes 117 Unipro UGENE Manual Version 1 20 0 e Navigation through an alignment e Optional coloring schemes for example Clustal Jalview like etc e Flexible zooming for large alignments e Export publication ready images of alignment e Several consensus calculation algorithms Using the Alignment Editor you can e Perform multiple sequence alignment using integrated MUSCLE and KAlign algorithms e Edit an alignment delete copy paste symbols sequences and subalignments e Build phylogenetic trees e Generate grid profiles e Build Hidden Markov Model profiles to use with HMM2 HMMsS tools Alignment Editor Components Here is the default layout of the editor UGENE COI m CON j File Acti
10. Use buttons e Invert selection to invert the selection of the sequences e Select all to select all sequences e Clear selection to clear the selection of all sequences The Add to project check box specifies to add the MSA file created from the subalignment to the active project Exporting Sequence from Alignment To export one sequence from an alignment select the sequence in the sequence list or in the sequence area and use the Export gt Save sequence context menu item The following dialog will appear m Export Selected Sequence from Alignment Export to file re feat to use Add document to the project Gap characters Keep E Trim Here it is possible to specify the result file location to select a sequence file format to define whether to keep or remove gaps chars in the sequence and optionally add the created document to the current project 132 Unipro UGENE Manual Version 1 20 0 Exporting Alignment as Image To export an alignment as image click the Export as image button on the editor toolbar or call the Export gt Export as image context menu item ETEY ET The Export Image dialog will appear where you should set name location export settings and format of the picture Alignment export settings neon meam i Indude sequences names Indude ruler Export to file UGENE supports export to the BMP JPEG JPG PNG PPM TIF TIFF XBM XPM and SVG im
11. You can turn off the Sequence offsets by unchecking the Actions View Show offsets main menu item or View Show offsets context menu item Navigation The Sequence area provides several flexible ways to navigate through an alignment The simplest way is to use the mouse and the scrollbars Alternatively you can use arrow keys on the keyboard to navigate The list of hot keys for quick navigation e PageUp to move one screen left e PageDown to move one screen right e Home to center the starting columns of the alignment e End to move to the trailing columns of the alignment 118 Unipro UGENE Manual Version 1 20 0 Hint if you use Shift key with the hot keys above you will navigate through the rows For example Shift PageDown will move one screen down Finally you can use the Go to position dialog from the Actions menu the context menu or the editor toolbar Enter the column number base coordinate and the view will be centered to the corresponding base Coloring Schemes There are various coloring schemes for DNA and amino alphabets available To change the scheme activate the Colors context menu el Go to position Add Copy Colors No colors Jalview Percentage Identity Highlighting Edit Align Percentage Identity gray Tree UGENE Statistics Custom schemes View Export or use the Highlighting tab of the Options Panel Talv
12. familiar with any programming language The workflow schemas comprise reproducible reusable and self documented research routines with a simple and unambiguous visual representation suitable for publications The workflow schemas can be run both locally and remotely either using graphical interface or launched from the command line The elements that a schema consists of corresponds to the bulk of algorithms integrated into UGENE Additionally you can create custom workflow elements U UGENE Workflow Designer Build HMM from alignment and test it AS File Actions Settings Tools Window Help OQOobhinS amp gt Ba 7 amp Itemstyle Runmode Scripting mode Elements Samples Data sources Ja Datasinks J Basic analysis tL Project Collocation search Extract annotated regions _ Find repeats ACS Find substrings O Import PHRED qualities l ai 7 Local BLAST search l ad oS Local BLAST search ORF marker gt Request to remote database xy Smith Waterman search EA Align with Clustalw K Align with Kalign E Align with MAFFT 1 Align with MUSCLE hn Align with T Coffee Tr iption factor Build frequency matrix a O zts Reads MSA s from HIV 1 aln Read sequence Reads sequence s from human T1 fa Write sequence s from For each sequence from Read sequence and set of annotations from HMM_ search
13. penalty for extending a gap Report results simple heuristic which allows to filter intersected hits If it is set to none the algorithm may report large set of almost identical results in the same region Minimal score another simple heuristic which measures sequences similarity It is more convenient than using some abstract scores If set to 100 the algorithm will search for exact substring match The results of the search are saved as annotations or as multiple alignment To set the saving parameters go to the Input and output tab of the dialog If you want to save the results as annotations input the annotations saving parameters Annotation name Group name Annotation type Description and a file to save the annotation to Also you can add qualifier with corresponding pattern subsequences to result annotations Check the corresponding checkbox for it If you want to save the results as multiple alignment select the following parameters Dy Smith Waterman Search Save results as Multiple alignment Aligner options Alignment files directory path Set advanced options Template for alignment files names PN _ SN _ c Template for reference subsequences names SN 5 E Template for pattern subsequences names PN 5 E Pattern sequence name Pi SN Reference sequence name prefix PN Pattern sequence name prefix S Subsequence start position E Subsequence end position L Subsequence length C Counter hms Time
14. 5 terminal ren h i H Unload selected documents Add tamer K D P T RR N l ACCCCACCCGTAGGTG E LL Li SF as i ke L 6 amp 10 12 14 16 156 20 22 24 CTTTCTGGGCTGGCCATCCAC F oI gt W R L H E L W G Y Ti Export Remove hz Save a copy STEMS Hame 9 NC 001363 Features murine gb The picture above illustrates an option to visualize the selected DNA sequence object using the Sequence View a complex and extensible Object View that focuses on visualization of sequence objects in combination with different kinds of related data sequence annotations graphs chromatograms sequence analysis algorithms Note that the Sequence View is described in more details in the separate document ation section Exporting Objects The document objects can be exported into a new document For more details see the following chapters Exporting Sequences to Sequence Format Exporting Sequences as Alignment Exporting Alignment to Sequence Format Exporting Nucleic Alignment to Amino Translation Export Sequences Associated with Annotation Exporting Sequences to Sequence Format Select a single or several sequence objects in the Project View window and click the Export Export sequences context menu item 2 Unipro UGENE Manual Version 1 20 0 Help ax Go Bi 4 Ga ga Ga Ge Ga Fea cor A i 85 SW wm FF i X Phaneropt O B S 0 E3 T E np 20 100
15. Assembly Browser Hotkeys e Assembly Overview Hotkeys e Reads Area Hotkeys Assembly Overview Hotkeys The following hotkeys are available for the Assembly Overview Hotkey Shift move mouse Ctrl wheel Alt click wheel move mouse Reads Area Hotkeys The following hotkeys are available for the Reads Area Hotkey wheel double click click move mouse arrow Ctrl arrow Page Up Page Down Home End Ctrl G Action Zoom the Assembly Overview to selection Zoom the Assembly Overview Zoom the Assembly Overview in 100x Move the Assembly Overview Action Zoom the Reads Area Zoom in the Reads Area Zoom in zoom out the Reads Area Move the Reads Area Move one base in the corresponding direction in the Reads Area Move one page in the corresponding direction in the Reads Area Move one page up down in the Reads Area Move to the beginning end of the assembly in the Reads Area Focus to the Go to position field on the toolbar 156 Phylogenetic Tree Viewer Unipro UGENE Manual Version 1 20 0 The Phylogenetic Tree Viewer is intended to display a phylogenetic tree built from an alignment or loaded from a file e g a Newick file UGENE COI m COT 1 File Actions Settings Tools Window Help loomana aa aSr yeg Tree view COl16 nwk 3 HO sophya_altaica_EF5 icolorana_bicolor_EF oeseliana_roeseli lontana_montana etrioptera_japonica ampsocleis_sedakov erac
16. File path Select file and file format and click on the OK button To import primer s click the Import primer s button The following dialog will appear 202 Unipro UGENE Manual Version 1 20 0 T Import Primers not Remove Add one or several files with primer sequences Note that all sequence formats supported by UGENE can be imported for example FASTA GenBank etc But the sequences must consist of ACGT characters only Click the mport button to import the added files into the primers library Secondary Structure Prediction The Secondary Structure Prediction plugin provides a set of algorithms for the protein secondary structure alpha helix beta sheet prediction from a raw sequence Currently available algorithms are e GORIV Jean Garnier Jean Francois Gibrat and Barry Robson GOR Method for Predicting Protein Secondary Structure from Amino Acid Sequence in Methods in Enzymology vol 266 pp 540 553 1996 Improved version of the GOR method in J Garnier D Osguthorpe and B Robson J Mol Biol vol 120 p 97 1978 e PsiPred Bryson K McGuffin LJ Marsden RL Ward JJ Sodhi JS amp Jones DT 2005 Protein structure prediction servers at University College London Nucl Acids Res 33 Web Server issue W36 38 Jones DT 1999 Protein secondary structure prediction based on position specific scoring matrices J Mol Biol 292 195 202 You can access these analysis capabilities
17. First lines to skip Lline s By pressing Preview one can bring up the view of the current annotations table which is produced from the input file with the specified parameters values The input file contents will also be shown at the bottom part of the dialog nents reve Raw file preview name start end quall a1 10 20 pp a1 19 40 ppe The preview table headline indicates the types of the information contained in the corresponding columns By default the values are ignored To specify a column role click on the corresponding headline element 17 Unipro UGENE Manual Version 1 20 0 UJ Select the Role of the Column Column role O Annotation start position Add offset 0 Annotation end position Indusive E Annotation length D Complement strand mark Mark value D Annotation name 0 Annotation group Qualifier Ignore this column The annotation start and end positions must be specified It is possible to add an offset to every read start position by checking the Add offset checkbox and to shorten annotations by one from the end by uncheking the nclusive checkbox When all the roles are specified press Run With the Add to project checkbox specified and a Sequence View opened on success you will see the Sequence View with annotations linked 14 4 6 68 10 12 14 16 16 20 4 44 2 46 30 2 M J J 40 42 CCAGATTCAGTTCCTTTAATAAAGAGATTAATTTCAATATTAA
18. If your Linux is not Ubuntu or Fedora then universal binary package is the only choice Otherwise for more tight integration with the systems you can install UGENE from corresponding repositories following these guides e Native installation on Ubuntu e Native installation on Fedora Please note that the repositories may be updated a little later the official UGENE release date Installation on Windows To install UGENE on Windows e Download UGENE Windows installation package ae Windows Packages for Windows XP Windows Vista Windows and higher Windows versions Installers e Download 32 bit Standard or Full installer package e Download 64 bit Standard or Full installer package ip bundels Download 32 bit portable Standard or Full zip bundle e Download 64 bit portable Standard or Full zip bundle e Download 64 bit NGS portable zip bundle caution zip bundle size is about 4Gb e Launch the downloaded exe le and follow the Unipro Setup wizard Unipro UGENE Manual Version 1 20 0 Welcome to the UGENE Setup Setup will guide you through the installation of UGENE It is recommended that you close all other applications before starting Setup This will make it possible to update relevant system files without having to reboot your computer Click Next to continue Be sure that you launch the installer with an administrative Windows account If you have a problem with installation try to d
19. Import as separate sequences Merge into a single sequence Number of unknown symbols V for nudeic ar A for amine between parts 10 bases Join into alignment Documents and objects options Create a subfolder for each document Available parameters are described below Process directories recursively if this option is checked the import procedure recreates the hierarchy of the imported directories and all their sub directories in the database Otherwise only the content of the directories specified for import is uploaded to the Destinati on folder without taking into account any sub directories Create a subfolder for each file if this option is checked for each file uploaded to the database a new folder is created having the same name as the file and the file content is placed in the folder Otherwise the file data are imported into the Destination folder 266 Unipro UGENE Manual Version 1 20 0 Import as separate sequences if this option is selected and an uploaded file contains several sequences they are represented by distinct sequence objects in the database after the import is done Merge into a single sequence if this option is selected and an uploaded file contains several sequences they are merged into a single sequence object in the database after the import is done Join into alignment if this option is selected and an uploaded file contains several sequences they are joined into a
20. Search for transcription factor binding sites TFBS with weight matrix and SITECON algorithms Aligning short reads with Bowtie Bowtie 2 BWA BWA SW and UGENE Genome Aligner Contig assembly with CAP3 Search for ORFs Cloning in silico 3D structure viewer for files in PDB and MMDB formats anaglyph view support Protein secondary structure prediction with GOR IV and PSIPRED algorithms HMMER2 and HMMER3 packages integration Building using integrated PHYLIP and MrBayes packages and viewing phylogenetic trees Local sequence alignment with optimized Smith Waterman algorithm Combining various algorithms into custom workflows with UGENE Workflow Designer Search for a pattern of various algorithms results in a nucleic acid sequence with UGENE Query Designer Visualization of next generation sequencing data BAM files using UGENE Assembly Browser PCR in silico Spade de novo assembler User Interface Visual and interactive genome browsing including circular plasmid view Multiple alignment editor Chromatograms visualization 3D viewer for files in PDB and MMDB formats with anaglyph stereo mode support Phylogenetic tree viewer Easy to use Workflow Designer for custom computational workflows Easy to use Query Designer for analyze a nucleotide sequence using different algorithms at the same time Assembly Browser for visualize and efficiently browsing large next generation sequence assemblies High Performance Computing Complete support o
21. Seed seed use lt int gt as the seed for pseudo random number generator The following flags are available No unpaired alignments no mixed by default when bowtie2 cannot find a concordant or discordant alignment for a pair it then tries to find alignments for the individual mates This option disables that behavior No discordant alignments no discordant by default bowtie2 looks for discordant alignments if it cannot find any concordant alignments A discordant alignment is an alignment where both mates align uniquely but that does not satisfy the paired end constraints This option disables that behavior No forward orientation nofw if nofw is specified bowtie2 will not attempt to align unpaired reads to the forward Watson reference strand No reverse complement orientation norc if norc Is specified bowtie2 will not attempt to align unpaired reads against the reverse complement Crick reference strand No overlapping mates no overlap if one mate alignment overlaps the other at all consider that to be non concordant No mates containing one another no contain if one mate alignment contains the other consider that to be non concordant Select the required parameters and press the Start button Building Index for Bowtie 2 To build Bowtie 2 index select the Tools Align to reference Build index item in the main menu The Build Index dialog appears Set the Align short
22. T Export Imag Export settings Indude position marker Indude selection marker File name jpg Format Width 1211px Height 371px Quality Here you can browse for the file name select the width height and resolution of the image as well as its format svg ps pdf bmp jpeg jog png ppm tif or tiff Also you can include position and selection markers to the image by the corresponding checkboxes Note that if a sequence file contains several sequences it is possible to view the circular views of the sequences in the same Circular Viewer area 1 ee a NP_597742 2 NP_597744 1 NP_597742 2 3 terminal repeat CVUS5 762 4733bp NC_001363 Itis possible to 5 terminal repeat resize the areas NP 040335 1 You can work with these circular views at the same time Circular View Settings To configure circular view settings go to the Circular View Settings tab in the Options Panel Activate the circular view for a sequence and the following settings will appear 88 Unipro UGENE Manual Version 1 20 0 Ruler Show ruler line Show coordinates Label fontsize 10 7 w Annotations In the title section you can show or hide title and length change font size and attribute In the ruler section you can show or hide ruler line and coordinates and change the label font size In the annotation section you can select the label position and change the label size The following label pos
23. each added short read is a small DNA sequence file At least one read should be added You can also configure other parameters They are the same as in the original Bowtie 2 you can read detailed description of the parameters on the Bowtie 2 manual page Select one of the following alignment modes The end to end alignment mode By default Bowtie 2 performs end to end read alignment That is it searches for alignments involving all of the read characters This is also called an untrimmea or unclipped alignment When the local option is specified Bowtie 2 performs local read alignment In this mode Bowtie 2 228 Unipro UGENE Manual Version 1 20 0 might trim or clio some read characters from one or both ends of the alignment if doing so maximizes the alignment score The following parameters are available Number of mismatches N sets the number of mismatches to allowed in a seed alignment during multiseed alignment Can be set to 0 or 1 Setting this higher makes alignment slower often much slower but increases sensitivity Seed length L Sets the length of the seed substrings to align during multiseed alignment Smaller values make alignment slower but more senstive Add columns to allow gaps dpad Pads dynamic programming problems by lt int gt columns on either side to allow gaps Disallow gaps gbar disallow gaps within lt int gt positions of the beginning or end of the read
24. 199945 MADZ71 1 pfm Direct strand 26 06o 1999359 199944 MADZ71 1 pfm Direct strand 14 07 199956 199945 Made 71 1 pFrm Direct strand 57 60 Also you can see the matrix by using the View matrix button O View Matrix 0 3 79 40 66 46 65 11 65 g4 735 4 3 1 2 5 2 3 1 0 3 4 1 0 5 3 2 2 19 11 50 29 4F 22 8 amp 1 1 4 I The regions found by the weight matrix algorithm can be saved as annotations to the DNA sequence in the Genbank format by pressing the Save as annotations button After saving the file with resulting annotations will be automatically added to the current project and the annotations will be added to the original sequence Note that in case of selecting JASPAR or UNIPROBE matrix the resulting annotations will contain the given matrix properties 246 Unipro UGENE Manual Version 1 20 0 amp Weight Matrix Search Save annotation s to Existing table Create new table Y Annotation parameters Group name lt auto gt Annotation name misc_feature Direct strand 85 35 _ Direct strand 85 75 i il a n See also e Searching JASPAR Database e Building New Matrix Searching JASPAR Database Press the Search JASPAR database button in the Weight matrix search dialog The following dialog will appear 247 Ka Weight Matrix Search bo wertebrates b urochordates 4 plants squamosa MA0082 1 myb Ph3 MA0054 1 idl MA0120 1 b IP911 b IP910
25. 59 549 26 mer C Mismatches 0 bp Y Reverse primer AAAAAACGTACGTCGT Tm 38 25 C 16 mer C E Mismatches Obp 3 perfect match 15 bp Maximum product 5000 bp Betract annotation Show primers details Warning Set dimer can be formed Delta G 15 4 kcal mole Base Pairs 11 There are the following parameters Forward primer forward primer Reverse primer on the opposite strand from the forward primer Mismatches mismatches limit 3 perfect match specify the number of nucleotides at the 3 end that must not have mismatches Maximum product maximum size of the amplified sequence Extract annotations specify the type of extracted annotations Inner All intersected or None e Value Inner is selected by default When this value is selected the extracted PCR product contains annotations from the original sequence located within the extracted region e Value All intersected specifies that all annotations of the original sequence that intersect the extracted region must be extracted as well e Value None specifies that annotations from the original sequence must not be extracted Choosing primers Type two primers for running In Silico PCR If the primers pair is invalid for running the PCR process then the warning is shown Also primer s for the running In silico PCR can be chosen from a primer library Click the following button to choose a primer from the primers library 198 Unipro UGENE Manual Ver
26. Creating Signals Generating Signals e Complex Signals Recognition on a Sequence e Shared Database e Configuring Database Connecting to a Shared Database Adding Data to the Database Database in the Project Deleting Data Drag n drop in the Database Exporting Objects from the Database e UGENE Public Storage e UGENE Command Line Interface e CLI Options e CLI Predefined Tasks e Format Converting Sequences e Converting MSA Extracting Sequence Finding ORFs Finding Repeats Finding Pattern Using Smith Waterman Algorithm Adding Phred Quality Scores to Sequence Local BLAST Search Local BLAST Search Remote NCBI BLAST and CDD Requests Annotating Sequence with UQL Schema Building Profile HMM Using HMMER2 Searching HMM Signals Using HMMER2 Aligning with MUSCLE Aligning with ClustalW Aligning with ClustalO Aligning with Kalign Aligning with MAFFT Aligning with T Coffee Building PFM Searching for TFBS with PFM Building PWM Searching for TFBS with Weight Matrices Building Statistical Profile for SITECON Searching for TFBS with SITECON Fetching Sequence from Remote Database Gene by Gene Report Reverse Complement Converting Sequences Variants Calling e Generating DNA Sequence e Creating Custom CLI Tasks e APPENDIXES e Appendix A Supported File Formats e Specific File Formats UGENE Native File Formats e Other File Formats e Tutorials e Using BioMart with UGENE Environment requirements Installing UGENE extension on Mozilla Fire
27. In the Annotation parameters group you can specify the name of the group and the name of the annotation If the group name is set to lt auto gt UGENE will use the group name as the name for the group You can use the characters in this field as a group name separator to create subgroups If the annotation name is set to by type UGENE will use the annotation type from the Annotation type t able as the name for the annotation Also you can add a description in the corresponding text field Select the parameters and click on the Save button The corresponding annotations will be saved Dotplot The Dotplot plugin provides a tool to build dotplots for DNA or RNA sequences This allows comparing these sequences graphically Using a dotplot you can easily identify such differences between sequences as mutations inversions insertions deletions and low complexity regions Also the plugin provides advanced features comparing multiple dotplots navigation in a dotplot dotplots synchronization saving and loading a dotplot etc An example of a dotplot view 106 Unipro UGENE Manual Version 1 20 0 1 10k 20k 30k 40k 30k 60k FOK 0k 90k 100k 110k 120k ae 426 10k 1 lt z oe tae r i Poe is Targ Cate 6 z ma y Ba ae 30k a TR 40k Ei 2 7 i D ima ri p 30k BJ w ene i a i ue a te E fai 7 3 Po Fa m FOK GOK ee a Ki 5y cn atch ou ee Re chy ae 3a Tors pelle 90k age 1 F
28. MDY Date Here you can select a file to save the alignment to Alignment files directory path parameter Using the Set advanced options checkbox you can select the saving options You can set the different templates for files names create your own or create by using the following E adds a subsequence end position hms adds a time MDY adds a date S adds a subsequence start position L adds a subsequence length SN adds a reference sequence name prefix PN adds a pattern sequence name prefix C adds a counter You can create templates for alignment files names reference subsequence names pattern subsequence names and for pattern sequence name 211 Unipro UGENE Manual Version 1 20 0 2 File Actions Settings Tools Window Help E ebiaaaeak wo HE Project Fa Name filter AT Consensus Objects Reference subsequence 4 i human_Tl fa 8 7 I HMM2 The HMM2 plugin is a toolkit based on the Sean Eddy s HMMER2 package While working on this plugin we were guided by the following principles e Make the HMMER2 tools accessible to a wider user audience by providing graphical interface for all supported utilities for most of the platforms e Be compatible with the original HMMER2 package e Create the high performance solution utilizing modern multi core processors and SIMD instructions The current version of UGENE provides user interface for three HMM2
29. Pa SOS conflict MRNA SON OA Os conflict AAB59342 1 SON A conflict mRNA Ss conflict A conflict we Fo Yoo misc_recomb mRNA we ra 5 Y conflict mRNA Fa mRNA mRNA AAB59341 1 The 3D Structure Viewer adds 3D visualization for PDB and MMDB files 80 Unipro UGENE Manual Version 1 20 0 8 30 Structure viewer Active view li 1v6C 2 m Display Links Add 36 J g5 100 105 q110 1145 120 125 130 135 140 145 150 155 160 165 PHONANIAHIVEY FH EACWGYS 7 SLY AATDT CVS CGANVVTHSLGOGSGSTITERNALNTHYNWNCVLLIAAACH ACD SS Y T 9 10 11 12 13 14 15 16 17 18 198 20 At Be 23 BA 235 26 BF 25 33 30 C A C G C G A T A G C A T T G C G A G A C G C T C A R D a qI A R RO OW 2 R z T L E R z I 12 4 6 amp 10 12 14 16 18 20 22 24 26 26 30 32 34 36 36 40 42 44 46 46 50 52 54 56 5660 CCACACGTGCCECTATCCTAACGCTCTGCGCACCTCEGCOCTCETGGGCATACAGCCETCATAGA ii H R C Q z WF z gt z C G I D Cc F R lt gt Name Value B gP Annotations MyDocument_9 gb E y misc_feature 0 1 O misc_feature 16 34 The DNA Graphs Package shows various graphs for sequences 81 Unipro UGENE Manual Version 1 20 0 09 AYO27935 standard sequence Me cz TE h e ae Y Informational Entropy 11514 19693 Window 500 Step 50 EO T E E E E E A E Se E E E E E E EE ET ed a eet cars tem cnet pe pn ec eee ena eine tse apes peter eee nee endat eae eee teens ate GO Content 3 11614 196935 Window 30 Step 10 are eee
30. Select an appropriate minimum and maximum value and click the OK button to show the graph of cutoffs The graph is divided into 2 parts The upper part shows values greater than the specified Maximum value The lower part of the graph shows values lower than the specified M inimum value For example Values greater than Maximum Values lower than Minimum _ Saving Graph Cuttoffs as Annotations To save graph cuttoffs as annotations select the Graph gt Save cuttoffs as annotations item in the graph contex menu The following dialog will appear 105 Unipro UGENE Manual Version 1 20 0 Py Save Graph Cutoffs as Annotations Maximum cutoff Minimum cutoff Area to annotate Around cutoff values Between cutoff values Y Save annotation s to Existing table 9 NC_014267 features NC_014267 1 gb Create new table Use auto annotations table Y Annotaton parameters Group name lt auto gt annotation type Annotation name graph_cutoffs Description The following parameters are available Maximum cutoff maximum cutoff value Minimum cutoff minimum cutoff value Around cutoff values saves the values around cutoffs values Between cutoff values saves the values between cutoffs values In the Save annotation s to group you can set up a file to store annotations It could be either an existing annotation table object a new annotation table or auto annotations table if it is available
31. This is an insensitive parameter Drop chain threshold D drop chains shorter than FLOAT fraction of the longest overlapping chain Rounds of mate rescues m perform at most INT rounds of mate rescues for each read Skip mate rescue S skip mate rescue Skip pairing P in the paired end mode perform SW to rescue missing hits only but do not try to find hits that fit a proper pair Score for a match A matching score Mismatch penalty B mismatch penalty The sequence error rate is approximately 75 exp log 4 B A Gap open penalty O gap open penalty Gap extention penalty E gap extension penalty A gap of length k costs O k E i e Gap open penalty is for opening a zero length gap Penalty for clipping L clipping penalty When performing SW extension BWA MEM keeps track of the best score reaching the end of query If this score is larger than the best SW score minus the clipping penalty clipping will not be applied Note that in this case the SAM AS tag reports the best SW score clipping penalty is not deducted Penalty unpaired U penalty for an unpaired read pair BWA MEM scores an unpaired read pair as scoreRead1 scoreRead2 NT a nd scores a paired as scoreRead1 scoreRead2 insertPenalty It compares these two scores to determine whether we should force pairing Score threshold T don t output alignment with score lower than score threshold This option only affects output S
32. b UGENE plugins have unified interface and work logic Also user who is already familiar with UGENE could cope with a new module faster Thus ExpertDiscovery uses reliable interface and visualization solutions Sequence view annotation view task manager etc of UGENE c Extension and combination of results possibilities appear For example ExpertDiscovery markups can be UGENE algorithms results SITECON Weight Matrix Query Designer etc d Data formats ExpertDiscovery can read sequences in any format which is supported by UGENE FASTA FASTAQ Genbank GFF EMBL etc To open the ExpertDixcovery go to the Tools gt Expert Discovery main menu item More detailed information about ExpertDiscovery you can find below Loading Sequences Mapping Sequences Markup Sequences Creating Signals Generating Signals Complex Signals Recognition on a Sequence Loading Sequences To load sequences to ExpertDiscovery click on the New ExpertDiscovery Document toolbar button m UGENE Expert Discovery I File Actions Settings Tools Window Help 868 2jba PAE F The following dialog will appear 258 Unipro UGENE Manual Version 1 20 0 m Load Positive and Negative Sequence Bases This is the first step of creating a new ExpertDiscovery project Load the sequences you want to analyze by choosing any file with a sequence or multiple sequences Positive sequence base contains a regulation object you are in
33. field Yes answer means that the gene is in the genome No answer MIGHT mean that there is no gene in the genome It is a good idea to analyze al the No sequences using annotated files Just open a file and find a sequence with a name of a gene that has No result Parameters in Input sequence file Url datasets final name Annotation name used to compare genes and reference genomes using blast_result by dafault String exist file lf a target report already exists you should specify how to handle that Merge two table in one Overwrite or Rename existing file using Merge by default String ident Identity between gene sequence length and annotation length in per cent BLAST identity if specified is checked after using 90 0 percents by default Number 286 Unipro UGENE Manual Version 1 20 0 out Output report file String blast out Location of BLAST output file String search type Type of BLAST searches using blastn by default String db name Name of BLAST DB String blast path Path to BLAST DB String expected value This setting specifies the statistical significance threshold for reporting matches against database sequences using 10 0 by default Number gapped aln Perform gapped alignment using use by default Boolean blast name Name for annotations using blast_result by default String tmpdir Directory for temporary files using UGENE temporary direct
34. format See also Assembly Browser ClustalW aln A multiple sequence alignments MSA file format See also Alignment Editor EBWT ebwt A Bowtie prebuilt index file See also Bowtie EMBL em emb embl A rich format for storing sequences and their annotations See also Sequence View 290 FASTA FASTQ Genbank GFF HMM MMDB MSF Mega Newick fa mpfa fna fsa fas fasta sef seqs fastq gb gbk gen genbank gff hmm prt msf meg meg gz nwk newick Unipro UGENE Manual Version 1 20 0 One of the oldest and simplest sequence file format See also Sequence View A file format used to store a sequence and its corresponding quality scores It was Originally developed at the Wellcome Trust Sanger Institute See also Sequence View A rich format for storing sequences and associated annotations See also Sequence View The Gene Finding Format GFF format is used to store features and annotations See also Sequence View A file format to store HMM profiles See also HMM2 HMM 3 ASN 1 format used by the Molecular Modeling Database MMDB See also 3D Structure Viewer A multiple sequence alignments file format See also Alignment Editor A multiple sequence alignments file format See also Alignment Editor A tree file format See also Building
35. ml BED or position list file String bg Per sample genotypes Boolean mC Mapping quality downgrading coefficient Number bT Pair trio calling String mB Disable BAQ computation Boolean me Gap extension error Number mE Extended BAQ computation Boolean bF Indicate PL Boolean vw Gap size Number m6 llumina 1 3 encoding Boolean bi INDEL to SNP Ratio Number bA Retain all possible alternate Boolean vD Max number of reads per input BAM Number md Max number of reads per input BAM Number mL Max INDEL depth Number va Alternate bases Number v2 BaseQ bias String vd Minimum read depth Number v4 End distance bias Number v3 MapQ bias Number Q Minimum RMS quality Number v7 Strand bias Number mQ Minimum base quality Number mq Minimum mapping quality Number bd Min samples fraction Number b1 N group 1 samples Number bU N permutations Number bG No genotype information Boolean ml No INDELs Boolean mo Gap open error Number mP List of platforms for indels String vp Log filtered Boolean bP Prior allele frequency spectrum String bQ QCALL likelihood Boolean mr Pileup region String bs List of samples String mh Homopolymer errors coefficient Number bt Mutation rate Number Unipro UGENE Manual Version 1 20 0 288 Unipro UGENE Manual Version 1 20 0 mA Count anomalous read p
36. mr I m If you want to see all annotation names click the Show all annotation names link The Previous annotation and Next annotation buttons seek to the previous or to the next annotation of the view correspondingly Find below information about annotations names properties that you can configure 71 e Annotations Color e Annotations Visability e Show on Translation e Captions on Annotations Annotations Color Unipro UGENE Manual Version 1 20 0 To change a color of all annotations of a certain type click on the corresponding color box in the annotations types table and select the required color in the appeared Select Color dialog Annotations Visability To show hide annotations with a certain name select this name in the annotations names table and check uncheck the Show annotations ch eck box below Another way to show hide the annotations is to select the Enable Disable highlighting item in the context menu of an annotation tz 1 Project T UGENE sars s NC_004718 File Actions Settings Tools Window Help he h t a Find qualifier Invert annotation selection Rename item F2 I Paste annotations Ctrl Shift V Go to position Ctrl 6 Select sequence region Ctrl A New annotation Ctrl N Copy Paste Select Add Analyze Align Cloning Fetch sequences from remote database Export Edit Show on
37. specify a custom region or search in the selected region Other Settings Other settings Remove overlapped results F Limit results number to 100000 This group contains additional common settings Remove overlapped results annotates only one of the overlapped results Limit results number to limits number of the searched results to the specified value 59 Unipro UGENE Manual Version 1 20 0 Annotations Settings 7 Save annotation s to Existing table NC_014267 features Create new table Y Annotaton parameters Group name auto gt Annotation type Misc Feature Description F Use pattern name In the Save annotation s to group you can set up a file to store annotations It could be either an existing annotation table object or a new annotation table In the Annotation parameters group you can specify the name of the group and the name of the annotation If the group name is set to lt auto gt UGENE will use the group name as the name for the group You can use the characters in this field as a group name separator to create subgroups If the annotation name is set to by type UGENE will use the annotation type from the Annotation type table as the name for the annotation Also you can add a description in the corresponding text field To use a pattern name for the annotations check the corresponding checkbox After that click the Create annotations button The
38. tmpadir directory for temporary files String Optional in semicolon separated list of input files String Required out output file String Required format format of the output file String Optional Example ugene align clustalw in COI aln out COI sto format stockholm Aligning with ClustalO Task Name align clustalo Create alignment with ClustalO ClustalO is a general purpose multiple sequence alignment program for proteins Q ClustalO is used as an external tool and must be installed on your system Parameters in Input alignment Url datasets format Document format of output alignment using clustal by default String out Output alignment String max guidetree iterations Maximum number guidetree iterations using 0 by default Number max hmm iterations Maximum number of HMM iterations using 0 by default Number iter Number of combined guide tree HMM iterations using 1 by default Number toolpath ClustalO location using the path specified in UGENE by default String auto Set options automatically might overwrite some of your options using False by default Boolean tmpdir Directory to store temporary files using UGENE temporary directory by default String Example ugene align clustalw in test aln out test_out aln format clustal Aligning with Kalign Task Name align kalign Multiple sequence alignment with Kalign Parameters
39. 1 a n 4 one eat a 3 els 4 ai 100k ways Jig Jr 110k i Po nee As ea oo oe ty ea on e ae Po oto Go ise Wiehe q boa i s4 Par L TE Bae ba ewan sone ee er een sO fee co fe ple tre NC_014267 sequence min length 11 identity 100 116k 2s ae iat Sy i The Dotplot plugin uses the Repeat Finder plugin to build a dotplot make sure you have the Repeat Finder plugin installed The Dotplot features are described in more details below Creating Dotplot Navigating in Dotplot Zooming to Selected Region Selecting Repeat Interpreting Dotplot Identifying Matches Mutations Invertions etc Editing Parameters Filtering Results Saving Dotplot as Image Saving and Loading Dotplot Building Dotplot for Currently Opened Sequence Comparing Several Dotplots Creating Dotplot To create a dotplot select the Tools Build dotplot main menu item The Build dotplot from sequences dialog will appear 107 Unipro UGENE Manual Version 1 20 0 File with first sequence E Join all sequences found in the file Compare sequence against itself File with second sequence E Join all sequences found in the file Here you should specify the File with first sequence Also you should either check the Compare sequence against itself option or select the Fi le with second sequence Optionally you can select to Join all sequences found in the file for the first and or for the second file If you select to
40. Aoo TT LT LT T i 1 500 1k 1 5k 2k 2 5k 3k 3 5k 4 4 5k 5k 5834 i Show circular view E yot Zoom buttons 5 HoT apture Graphs c Show complement strand 6ra ot uw deeeene 73 gt E D _ Automatiojannotatiens highlighting ef z 7 p Show amino translations _ Lb C L ar is Tougle L ccACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCEATTIGTCTAGTGT 44 645 650 655 660 665 670 675 680 685 690 596 GAT GGCCCCTCCATTCGCACCECCTCCTTGAATAGACACAGACAGLCTAACAGATCACS Amino transtation _ Select sequence region See also e Toggling Views e Capturing Screenshot e Zooming Sequence e Showing and Hiding Translations e Selecting Sequence Sequence Overview The Sequence overview is an area of the Sequence View below the sequence toolbar It shows the sequence in whole and provides handy navigation in the Sequence zoom view and the Sequence details view amp NC_001363 dna OR Olas 3 8k 4375 3 6k 3 4k 3k 2 6k 2656 mr 2 4K 2 2k 17 bp Mw 4 Scrolls the sequence details view Scrolls the sequence zoom view CC eee te lt a Sn ai ta aat a a j ai T E T l gt R 1 R 3 R 3 A i F F H E z f 1 CCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTT Lk ee aea EE ra E eS EE e E a S R a E a iGGCGCTCTGCCGTGGAAATTGGCTCTGGAGTAGTGGGTCCAATTCTAGTTCCAGAA R js E RS OV AG UE UV OSV UE UD GON UU DD M L When the sigma butto
41. Consensus type specifies the method to build the consensus tree Select one of the following e Strict specifies that a set of species must appear in all input trees to be included in the strict consensus tree e Majority Rule extended specifies that any set of species that appears in more than 50 of the trees is included The program then considers the other sets of species in order of the frequency with which they have appeared adding to the consensus tree any which are compatible with it until the tree is fully resolved This is the default setting e M1 includes in the consensus tree any sets of species that occur among the input trees more than a specified fraction of the time see the Fraction parameter below The Strict consensus and the Majority Rule consensus are extreme cases of the MI consensus being for fractions of 1 and 0 5 respectively e Majority Rule specifies that a set of species is included in the consensus tree if it is present in more than half of the input trees Fraction becomes available when the Consensus type parameter is set to M1 Specifies the fraction Display tree in new window displays tree in new window Display tree with alignment editor displays tree with alignment editor Synchronize alignment with tree synchronize alignment and tree Save tree to file to save the tree built Press the Build button to build a tree with the parameters selected MrBayes The Building Ph
42. GOK p a 547 Lat pene ee ete ee ali Bree arg ae a ga R Pe E ge Ay bet a a a co UOT ORS tr aa H Lie ise ate eT E Ala pa eae Cr ata Dee ty WW ag rie Se eta So Ue oat z i 5 at oo Ao Se ty rW oT n Li Lo oe a a on aie me na m s ome i ri J AH eel oe i mae t ih eT i r E ea ee EHI E aE ee ae ee Pa id Ee a a8 i ier 6 ee eee aoe He d r Pa at ara ani iie A Sh at a i eee ea bag ie iyi one ee KA RTE HEET EE Nal E E E ET LR ers TE EAE EET EEN fat ak eps epee n Pao oN ooh Bp Wide el oe ieee Poe oye oe wot pgs Rede Sea tea eR ee mad 90k os ol Oe nee Bo Ea ET HF Ger ie e ag ida TARAN wed oa oe th pe H N a Se eres ca eee 3 Ip taser od Po pe co aE renee ore Li ks ae ATje e aki lz a he Fa cet tS on G ae are k k Sa 4 J a arn gt sa a Ae o Bolg peo oe a a a anA Oe oon oe So A ee V eo oO Ta Er T Teee e ea ooo e cep o a ee EEE LIET DEIT Aan Amed oa PE r a et ec UY bg ah p ine nA sk ee cae vl ae te ratoe SEa B ole it i ee a Ts ek sete ey h fe ee bade Pedic TOR ae DA Tae E Ten bee viata Tie ip T OLAS ee i X seal 4 E da Et one a ere eel Re oa pi ar IE eS fede al rl as i SIAT ir aora Voy P ba a hyp pete PT Te dd m rigi 25 rm ty A rin Aeara a ts tele SRD cy ua ay eA cti a a a ee oe k D 1 00k 1 l i Hanes ie mas n yet F ne m ae HARYA abe ha uo ieee oe Fao z J we tle pe eae 1 gt k Jy oo mega aki 1 a aan a a Th tr aS Tiri el oo oP on aoe A UE Ean oi
43. Hetrodes_pupus EF540832 Swap Siblin P P l Mecopoda_elongata _Ishigaki __ Reroot tree NEER I z Mecopoda_elongata Sumatra_ Branch Settings aMecopoda sp Malaysia_ Export Tree Image Isophya_altaica EF540820 0 103 Phaneroptera_ falcata To show the collapsed clade select the Expand item in the node s context menu Swapping Siblings To rearrange two branches of an internal node select the Swap Siblings item in the node context menu or click the Swap Siblings button on the tree toolbar while the node is selected COll 6 nwk X Metrioptera_japonica_EF 540851 eBicolorana bicolor EF540830 Roeseliana_roeseli Montana_montana Gampsocleis_sedakovii_EF540828 Reroot tree fychia_baranovi Collapse Deracantha_deracantoides EF540 er m E r e i ie Export Tree Image Conocephalus_percaudata Conocephalus_ discolor Conocephalus_ sp 162 Unipro UGENE Manual Version 1 20 0 Zooming Clade Additionally to other zooming options you can use the Zoom In item in the context menu of the root node of a clade Adjusting Clade Settings When a clade is selected the branch and the labels formatting settigns are applied to the clade only Note that the settings are not applied to the collapsed branches if any See an example of changing branch settings for a clade Hicolorana bicolor EF SEO i ial mni ll Rneselians_roeseli Montana_mentana Metrioptera_jap
44. MUSCLE vode musaca E o Mode details The default settings are designed to give the best accuracy Command line muscle Advanced options Do not re arrange sequences stable ry Max iterations F Max time minutes Translate to amino when aligning Translation table 1 The Standard Genetic Code Region to align Whole alignment Column range By default UGENE does not rearrange sequence order in an alignment but the original MUSCLE package does To enable sequence rearrangement uncheck the Do not re arrange sequences stable option in the dialog One of the improvements to the original MUSCLE package is the ability to align only a part of the model When the Column range item is selected the region of the specified columns is only passed to the MUSCLE alignment engine The resulted alignment is inserted into the original one with gaps added or removed on the region boundaries To visually select the column range to align make a selection in the alignment editor first Then invoke the MUSCLE plugin Its column range boundary values will automatically match the given selection Aligning Profile to Profile with MUSCLE The Align Align profile to profile with MUSCLE context menu item allows to align an existing profile to an active alignment During this process the MUSCLE does not realign the profiles but inserts columns with gaps characters only characters For example the alignment in the picture belo
45. O pattern 5293 5305 Editing Sequence If the document is not locked it is possible to edit the sequence Edit sequence i Insert subsequence Ctrl I Remove Replace subsequence Ctri R A Rulers 5 Remove subsequence Reverse complement sequence ih Statistics L i Reverse sequence Complement sequence The Edit sequence submenu is available in the Actions main menu and in the Sequence View context menu Also you can use the corresponding shortcuts When you press the Ctrl I shortcut or select the Insert subsequence context menu item the following dialog is opened 61 Unipro UGENE Manual Version 1 20 0 4 Insert Sequence Paste data here Annotations region resolving mode Position to insert Expand affected annotation 7 Remove affected annotation gt Split Goin annotation parts i Selection Split separate annotations parts before 7 39 Recalculate values of qualifiers Save to new file Merge annotations to this file Document location Document format FASTA Description of the dialog parameters Paste data here you must input the inserted subsequence This parameter is mandatory Annotated regions resolving mode defines either to Expand affected annotation Remove affected annotation Split join annotation parts or Split separate annotation parts in case when the subsequence is inserted to the sequence position where some annotations are presented Recalculate
46. Open a multiple sequence alignment file and select the Align with ClustalW item in the context menu or in the Actions main menu The Align with ClustalW dialog appears see below where you can adjust the following parameters Gap opening penalty cost of opening up a new gap in the alignment Increasing this value will make gaps less frequent Gap extension penalty cost of every item in a gap Increasing this value will make gaps shorter Weight matrix specifies a single weight matrix for nucleotide sequences or series of matrices for protein sequences For nucleotide sequences the weight matrix selected defines the scores assigned to matches and mismatches including IUB ambiguity codes it can take values e UB default scoring matrix used by BESTFIT for the comparison of nucleic acid sequences X s and N s are treated as matches to any IUB ambiguity symbol All matches score 1 9 all mismatches for IUB symbols score 0 e CLUSTALW previous system used by ClustalW in which matches score 1 0 and mismatches score 0 All matches for IUB symbols also score 0 For protein sequences it describes the similarity of each amino acid to each other The following values are available e BLOSUM BLOcks of Amino Acid SUbstitution Matrices first introduced in a paper by Henikoff and Henikoff These matrices appear to be the best available for carrying out data base similarity homology searches e PAM Point Accepted Mutation matri
47. Phylogenetic Tree Phyl ogenetic Tree Viewer 291 Nexus PDB pDRAW32 PFM Phylip PWM Raw SAM SCF SITECON nex nxs pdb pdw pfm phy pwm seq sam SCf sitecon Unipro UGENE Manual Version 1 20 0 A multiple alignment and phylogenetic trees file format See also Alignment Editor Building Phylogenetic Tree Phyl ogenetic Tree Viewer The Protein Data Bank PDB format allows to view the 3D structure of the sequence See also 3D Structure Viewer A sequence file format used by pDRAW32 software See also Sequence View A file format for a position frequency matrix See also Weight Matrix A multiple alignment file format See also Alignment Editor A file format for a position weight matrix See also Weight Matrix A raw sequence format See also Sequence View The Sequence Alignment Map SAM format is a generic alignment format for storing read alignments against reference sequences See also Assembly Browser Bowtie UGEN E Genome Aligner It is a Standard Chromatogram Format See also Chromatogra m Viewer A file format to store TFBS profile See also S TECON 292 Stockholm Swiss Prot Vector NTI Sequence VCF Sto txt Sw gb gp vct UGENE Native File Formats File format Dotplot UGENE database file Short Reads FASTA UGENE Workflow Designer schema File extension
48. Read sequence in Genbank format to Property Editor Element name HMM search HMM search Searches each input sequence for significantly similar sequence matches to all specified HMM profiles In case several profiles were supplied searches with all profiles one by one and outputs united set of annotations for each sequence To configure the parameters of the element go to Parameters area below Iterations Cc Parameters Filter by high E value 1 Filter by low score Number of segs Input data Output data Filter by high E value E value filtering can be used to exclude low probability hits from result 166 Unipro UGENE Manual Version 1 20 0 To learn more about the Workflow Designer read the Workflow Designer Manual follow the link on the UGENE documentation page DNA Annotator The DNA Annotator plugin provides an algorithm to search for sequence regions that contain a predefined set of annotations Usage example Open the Sequence View for a sequence that has annotations A good candidate here could be any file in Genbank format with a rich set of annotations Select the Analyze Find annotated regions item in the context menu The dialog will appear T Find Groups of Annotated Regions O cps 444 1443 misc_feature P omar lt lt click button to add new annotation gt gt Annotations to search Results Region si
49. Rue een A a a APET L wr T 1 r m fai DE ea Boy H o Ei I poo m ams te ot ee 7 aT ae Te ata dan e o E a eae Fae a atare ela g AA aE i e a Ea a a o a bepa aae ep a GOK 90K 100k 110k a AEO a a AE 116k NC_014267 sequence min length 11 identity 100 3 Inverted repeats The Dotplot plugin allows to search for inverted repeats as well Inverted repeats are shown contrary to the direct repeats Use the Search direct repeats and Search inverted repeats options of the Dotplot parameters dialog to select which repeats to draw the dialog is described here 439 445 450 455 460 465 470 475 460 465 490 495 300 505 450 455 450 470 475 E 4 Low complexity regions A low complexity region is a region produced by redundancy in a particular part of the sequence It is represented on a plot as a rectangular area filled with the matches 112 Unipro UGENE Manual Version 1 20 0 230 240 290 2iQ 280 290 300 310 320 330 340 347 7 dbo gt m NO No agnas s TA Ssni 7 A Hint Compare sequence with itself to easily find low complexity regions in it Editing Parameters It is possible to edit parameters of a built dotplot Right click on the dotplot and select the Dotplot Parameters context menu item 5 ME k Parameters i F ge l _ amp amp Go to position Col 6 Save Load 2 4E Select sequence region Crl A R
50. Show scores for symbols not used in alignment Skip gaps in consensus position increments Save profile to file File Hypertext HTML Comma separated CSV Here is a brief description of the options that can be set in the dialog Profile mode Counts Percents select the Percents to have scores shown as percents in the report Show scores for gaps check this item if you want gap characters statistics to be shown in the report Show scores for symbols not used in alignment if a symbol is not used in the alignment at all it won t be shown in the report Check this item to make all symbols of alignment alphabet reported Skip gaps in consensus position increments consensus ruler configuration If checked the gaps in consensus will not lead to ruler increments Save profile to file allows to save profile to a file in the HTML or CSV format The CSV format is convenient for further processing in worksheets editors like Excel The result profile in the HTML mode 170 Unipro UGENE Manual Version 1 20 0 U UGENE File Actions Settings Tools Window Help Oo e U COICOI Consensus Multiple Sequence Alignment Grid Profile Phaneropte fsophya_ai Bicolorana_ Roeseliana Montana n kamm Alignment file C Program Files Unipro UGENE data samples CLUSTALW COI aln CoOl Table content symbol counts Legend 10 25 50 70 SO No active tasks amp DNA Generator
51. The following dialog appears 223 Unipro UGENE Manual Version 1 20 0 T Align with MAFFT Input file Output file Advanced options 7 Gap opening penalty Offset works like gap extension penalty 0 00 E Maximum number of iterative refinement 0 The following parameters are available Gap opening penalty Gap opening penalty at group to group alignment Offset works like gap extension penalty offset value which works like gap extension penalty for group to group alignment Maximum number of iterative refine specifies the number of cycles of iterative refinement to perform T Coffee T Coffee is a multiple sequence alignment package T Coffee home page T Coffee To make 7 Coffee available from UGENE see the External Tools To use T Coffee open a multiple sequence alignment file and select the Align with T Coffee item in the context menu or in the Actions main menu The following dialog appears I Align with T Coffee Input file Output file Advanced options Gap opening penalty Gap extension penalty Number of iterations The following parameters are available Gap opening penalty indicates the penalty applied for opening a gap The penalty must be negative Gap extension penalty indicates the penalty applied for extending a gap Number of iterations specifies the number of iterations Bowtie Bowtie is a popular short read aligner Click this link to open Bowti
52. Translation Remowe Rulers This option is available for nucleotide sequences only It specifies to show the annotation on the corresponding amino sequence instead of the original nucleotide sequence in the Sequence Detailed View for example R E T K V E LCATTCACCAAAGTIGCAAA pp gt amp 10 12 14 16 18 230 22 24 TOTAAGTGCOTTTCAACTTT r W L E F z E G E N F You can enable disable this option by checking unchecking the Show on translation checkbox Captions on Annotations 72 Unipro UGENE Manual Version 1 20 0 It is possible to show a value of a qualifier of an annotation instead of the annotation type name in the Sequence Zoom View To enable this option for an annotation type check the Show value of qualifier check box and input the values of the required qualifiers in the text field nearby this check box See the image below cows O wm 0 Cees bh wa g Annotations Highlighting 1 500 k 15k 2k 25k 3k 35k 4k 45k Sk aes E er pee eee a een oo Annotation Color ey 5245 53k 5350 S4igf 5450 55k 5550 56k 5850 57k 5750 5833 aes 589 bp _ Baal r m T Baen E _A KF 0 D IT G6 RK EF 4b PR OL Baa E ik l DS B L SacI E 5445 5450 S455 5460 5465 5470 5475 5480 5488 comment mE fol CGETTITETCCTATAGACACCATICGTCAAGGACGGGGCCGAGG pic feature o Se i i al i E EE a a ne z S E 8A GLL ALLRI E F L I D T L Li L FE t FE SO
53. User Interface applied after restart a Directories Logging l Alignment Color Scheme j aiga External Tools WindowsVista Genome Aligner Workflow Designer Window Layout OpenCL Multiple documents Tabbed documents Project Open last project at startup Ask to save new projectonexit Statistical reports Enable statistical reports collecting Default settings C Reset settings to default on the next run The following settings are available on the tab Language of User Interface applied after restart here you can select UGENE localization Currently available localizations are EN and RU The default value Autodetection specifies that UGENE should use the operating system regional options to select the localization This setting is applied only after UGENE is reopened Appearance defines the appearance of the application Window Layout this option allows to control the behavior of windows multiple or tabs Open last project at startup if the option is checked the last project is opened when UGENE is started Also you can choose default settings for saving project Enable statistical reports collecting collects information about UGENE usage and sends it to the UGENE team to help improve the application i The collected information includes 1 System info UGENE version OS name Qt version etc 2 Counters info number of launches of certain tasks e g HMM search MUSCLE align The collect
54. WNC 014267 1 gb PBRS22 gb sars gb WJ PI The following parameters are available Separate sequence mode opens the sequences as separate sequences Merge sequence mode merges sequences into one sequence with selected number of unknown symbols between sequences Join sequences into alignment joins sequences into alignment Save document save document to the selected document Also you can change the order of the sequences by up and down arrows Choose the parameters and click the Open button Sequences will be opened in the selected mode In the Separate sequence mode sequences will be opened as separate sequences in selected order You can change the sequences order by drag and drop in the sequence view Annotations Editor The Annotations editor contains tools to manipulate annotations for a sequence It provides a convenient way to organize view and modify a single annotation as well as annotation groups An annotation for a sequence consists of e Name or key indicates the biological nature of the annotated feature e Location coordinates in the sequence e The list of qualifiers qualifiers are the general mechanism for supplying information about annotation Qualifiers are stored as pairs of name value strings Below is the default layout of the Annotations editor with an extra column for the note qualifier added 66 Unipro UGENE Manual Version 1 20 0 murine NC_001363 sequence p D
55. abi4 TOALA PEND collection comment Unipro UGENE Manual Version 1 20 0 Other Alpha He Helix Turn Helix Zinc coordinat Zipper Type Zipper Type tipper Type Zipper Type Zinc coordinat iat Ca a AAC18941 inc coordinating CORE MADS Myb BetaBetaAlpha zinc finge Leucine Zipper Leucine Zipper AP MBD like Leucine Zipper Leucine Zipper Here the matrices are divided into categories and you can read detailed information of a matrix which is represented by its properties It could help you to choose the matrix properly i The matrices provided with UGENE are located in the UGENE data position_weight_matrix folder Building New Matrix To create a position weight or frequency matrix from an alignment or a file with several sequences press the Build new matrix button in the Weight matrix search dialog or select the Tools Weight matrix Build weight matrix program main menu item 248 Unipro UGENE Manual Version 1 20 0 Help Create index File DNA Assembly Weight matrix HMMER2 tools d SITECOM HMMER 3 tools workflow Designer The Build weight or frequency matrix dialog will appear Input fle Output file Statistic options Statistic type Matrix options Matrix type Frequency matrix Build weight matrix Weight algorithm Berg and von Hippel The following parameters are available Input file an al
56. advanced parameters T Contig Assembly with CAP3 Advanced Clipping for poor regions Length and percent identity of an overlap Base quality cutoff for dipping c h2 Overlap length cutoff o Clipping range y Overlap percent identity cutoff p Quality difference score of an overlap Other parameters Max number of word matches t Base quality cutoff for differences b 20 l Band expansion size a Max gscore sum at differences d Max gap length in any overlap f Assembly reverse reads Similarity score of an overlap Match score factor m 2 Mismatch score factor n 5 Gap penalty factor g 6 Overlap similarity score cutoff s 900 Clipping for poor regions parameters Clipping of a poor end region of a read is controlled by parameters Base quality cutoff for clipping c the specified value should be more than 5 and Clipping range y the specified value should be more than 5 Quality difference score of an overlap parameters Base quality cutoff for differences b if an overlap contains a difference at bases of quality values q1 and q2 then the score at the difference is max 0 min q1 q2 b where b is the specified value The specified value should be more than 15 The difference score of an overlap is the sum of scores at each difference Max qscore sum at differences d remove an overlap if its difference score is greater than the specified value The specified value
57. ae es ee E E head a can ee Nig ee leg a eee ae ela E EA sai W614 12 5k 15k 13 5k 14k 145k 15k 155k 16k 16 5k 17k Tk ook 18 5k 19k 19693 z z P A E F T L E I Y F z Y R F 2 NH CTICCICCCCCOCCOAATTTACGOGTTAAAAATITGOGTGOGTTTTTTCAGCGTTAGATTTCAAAATA r t t S H A re S E S a gt S E S E E E S E E 12 4 6 amp 10 12 14 16 16 20 22 24 26 28 30 32 34 36 so 40 42 44 46 45 50 52 54 56 5860 gj gt Mame value E cy Y027935 standard features Haemophilus emb The Dotplot provides a tool to build dotplots for DNA or RNA sequences 82 Unipro UGENE Manual Version 1 20 0 20k 30k 40k 30k 60k FOK 0k 90k 100k 110k 120k 140 426 z ae i 2 T de Bh a a T ae a E E a o pen wit gt Dep Jim IA p TE c diao 5 is ec ee i TE Baie oo see ace Eua Gee rie ates pt wa an Et 2 i aauanbas Serio ON GOK 90k ru H z Bes 100k igs oa Bae aI 1 a Hie it ee 110k I ea i i ee ee tee 6 eae un nd es al con tn an eis Soe NC_014267 sequence min length 11 identity 100 A number of other instruments add graphical interface for popular sequence analysis methods 83 Unipro UGENE Manual Version 1 20 0 U UGENE murine NC_001363 sequence W File Actions Settings Tools Window Help 22 ff mi lf am mem 9 Ne 001363 sequence dna A Oo 3 5k 4k aK P 597742 2AP STT Ctrl 6 e Goto position Vo Select sequence region Crl 4 Ctrl
58. annotations will be created Also you can see the result statistic and navigation under the Search for field Results 1 1 Previous Next Searching for one or several patterns and names of the result annotations If you search for one pattern only than input the required name into the Annotation name field and leave the Use pattern name check box unchecked You can also search for several patterns at a time by e Inputting several patterns into the search field click lt Ctrl gt lt Enter gt keys to insert to a new line Search in Sequence Search for TGGCAAGCTAGC TITGCAAGGCATG e Inputting several patterns into the search filed in FASTA format 60 Unipro UGENE Manual Version 1 20 0 Search in Sequence Search for gt pattern 1 TGGCAAGCTAGC gt patternz TITGCAAGGCATG e Loading patterns from a FASTA file Even when you search for several patterns names of the found annotations will be identical by default the name is specified in the Annotatio n name field If you want to assign different names to annotations found for different patterns than you should e Input the patterns in FASTA format the latter two cases above e Check the Use pattern name checkbox in the Annotation parameters group Here is an example of the found annotations in the Annotations Editor sg Annotations MyDocument_l gb a Q misc feature 0 4 gt E patternl 74 35 gt E patternl 5267 5278 gt pattern 50 57
59. can Exclude gaps Show group statistics of multiple alignment shows group statistics when the collapsing is switched on Save profile to file allows to save profile to a file in the HTML or CSV format The CSV format is convenient for further processing in worksheets editors like Excel The result profile in the HTML mode 134 Unipro UGENE Manual Version 1 20 0 lily Distance matrix for COI Table content Hamming dissimilari Hetrodes pupus EF540832 Legend D0 25 50 70 90 Grid Profile Using the Alignment Editor you can create a statistic profile of a multiple sequence alignment The alignment grid profile shows positional amino acid or nucleotide counts highlighted according to the frequency of symbols in a row To create a grid profile use the Statistics Generate grid profile item in the Actions main menu or in the context menu To learn more about this feature refer to the DNA Statistics plugin documentation Advanced Functions This chapter is devoted to the advanced functions of the Alignment Editor You will learn how to build a grid profile export a picture of an alignment and build HMM profiles e Building HMM Profile Building HMM Profile The editor has capabilities to build a Hidden Markov Model profile based on the multiple sequence alignment This functionality is based on the Sean Eddy s HMMER package To build a HMM profile select the Advanced Build HMMER2 profile or the Advanced Bu
60. corresponding nucleotide in a window 103 Unipro UGENE Manual Version 1 20 0 e DNA Flexibility searches for regions of high DNA helix flexibility in a DNA sequence The average Threshold in a window is calculated by the following formula sum of flexibility angles in the window the window size 1 For more detailed information see DNA Flexibility paragraph e GC Content shows the percentage of nitrogenous bases either guanine or cytosine on a DNA molecule It is calculated by the following formula G C A G C T 100 e AG Content shows the percentage of nitrogenous bases either adenine or guanine on a DNA molecule It is calculated by the following formula A G A G C T 100 e GC Frame Plot this graph is similar to the GC content graph but shows the GC content of the first second and third position independently It is most effective in organisms with GC rich genomic sequence but it also works on all microbial sequences e GC Deviation G C G C shows the difference between the QG content of the forward strand and the reverse strand GC Deviation is calculated by the following formula C C 7 GFC e AT Deviation A T A T shows the difference between the A content of the forward strand and the reverse strand AT Deviation is calculated by the following formula A T At T e Karlin Signature Difference dinucleotide absolute relative abundance differe
61. during cloning On the Output tab of the dialog you can select the file to save the new molecule to As soon as the required parameters are selected press the OK button The fragments will be saved as annotations Also all the generated fragments are available in the task report 193 Unipro UGENE Manual Version 1 20 0 W Task report DigestSeque mesa Task report DigestSequenceTask status Finished tinte 0 00 00 023 Digest into fragments CVU55762 gb circular Generated 3 fragments i From Dral 1460 To Dral 3901 2442 bp 2 From Dral 3902 To Dral 3920 19 bp 3 From Dral 3921 To Dral 1459 2272 bp Refer to Notifications to learn more about task reports Creating Fragment To create a DNA fragment from a sequence region activate the Sequence View window and select either the Actions Cloning Create Fragment item in the main menu or the Cloning Create Fragment item in the context menu The Create DNA Fragment dialog appears m Create DNA Fragment Fragment Options E Indude Left Overhang E Indude Right Overhang Direct Reverse complement Direct Reverse complement If a region has been selected you can choose to create the fragment from this region Otherwise you can either choose to create the fragment from the whole sequence or choose the Custom item and input the custom region To add a 5 overhang to the direct strand check the nclude Left Overhang check box and input the required
62. factor 1 Upstream stimulatory factors Is a protein that in humans is encoded by the YY1 gene Description N acetylgalactosamine repressor AgaR negatively controls the expression of the aga gene cluster AgaC is the Enzyme IIC domain of a predicted N acetylgalactosamine transporting PEP dependent phosphotransferase system ArcA transcriptional dual regulator ArgR complexed with L arginine represses the transcription of several genes involved in biosynthesis and transport of arginine transport of histidine and its own synthesis and activates genes for arginine catabolism DNA binding response regulator in two component regulatory system with CpxA cAMP receptor protein Cysteine B Cytidine Regulator Deoxyribose Regulator DnaA is the linchpin element in the initiation of DNA replication in E coli Fatty acid degradation Regulon Factor for inversion stimulation Operon that encodes two transcriptional regulators FNR is the primary transcriptional regulator that mediates the transition from aerobic to anaerobic growth through the regulation of hundreds of genes Fructose repressor Ferric Uptake Regulation Galactose repressor Galactose isorepressor sn Glycerol 3 phosphate repressor Is a member of the GntP family transporters Histone like nucleoid structuring protein Isocitrate lyase Regulator Integration host factor lron sulfur cluster Regulator 1 lron sulfur cluster Regulator 3 LexA represses the transcription
63. files By default the path specified in the Application Settings is applied String Optional Default default in semicolon separated list of input sequence files String Required dbpath path to the BLAST database files String Required dbname base name of the BLAST database files String Required out output Genbank file the results of the search are stored as annotations String Required name name of the annotations String Optional Default blast result p type of the BLAST search String Optional Default blastn The following values are available e blastn e blastp e blastx e tblastn e tblastx e expectation value threshold Number Optional Default 10 Example 2 8 Unipro UGENE Manual Version 1 20 0 ugene local blast in input fa dbpath dbname mydb out output gb Remote NCBI BLAST and CDD Requests Task Name remote request Performs remote requests to the NCBI Saves the results as annotations Parameters in semicolon separated list of input files A file can be of any format containing sequences or alignments String Required db database to search in String Optional Default ncbi blastn The following databases are available e ncbi blastn for nucleotide sequences e ncbi cdd for amino acid sequences e ncbi blastp for amino acid sequences out output Genbank file String Required eval speci
64. for a protein sequence using the Analyze Predict secondary structure context menu item The dialog will appear 203 Unipro UGENE Manual Version 1 20 0 A Secondary Structure Prediction It supports the following options Algorithm you can choose the preferred algorithm Currently GORIV and PsiPred algorithms are available Range start Range end select the sequence range for prediction Results visual representation of the prediction results for example 204 Unipro UGENE Manual Version 1 20 0 Dy Secondary Structure Prediction Algorithm Range Start 1 Results Region gorlV_results 2 44 49 gorlV_results 3 61 68 gorlV_results Total predicted 3 Save as annotation select this button to save the results as annotations of the current protein sequence SITECON SITECON is a program package for recognition of potential transcription factor binding sites basing on the data about conservative conformational and physicochemical properties revealed on the basis of the binding sites sets analysis To cite SITECON use the following article Oshchepkov D Y Vityaev E E Grigorovich D A Ignatieva E V Khlebodarova T M SITECON a tool for detecting conservative conformational and physicochemical properties in transcription factor binding site alignments and for siterecognition Nucleic Acids Res 2004 Jul 1 32 Web Server issue W208 12 UGENE version o
65. for every column it selects the rarest symbol in the whole alignment with percentage in the column greater or equals to the threshold value e Strict the algorithm returns gap character if symbol frequency in a column is lower than the threshold specified 123 Unipro UGENE Manual Version 1 20 0 Also the General tab shows the general information about an alignment and allows to select a reference sequence The following chapter describes how to export a consensus sequence e Export Consensus Export Consensus To export consensus sequence use the Exprot consensus tab of the Options Panel Export to file The following parameters are available Export to file here you need to select path for the output file File format format for the output file When you click on the Export button the consensus sequence will be exported into selected output file Alignment Overview The alignment overview is shown automatically in the Alignment Editor To close the overview click on the Overview toolbar button To show the simple alignment overview use the Show simple overview context menu item of the overview v Show simple overview ii Calculation method ij IF H til b Display settings The following settings of the alignment overview are available Export as image you can export multiple alignment overview and simple alignment overview as image Use this context menu item to do it In the follow
66. for the sequences Markup is an annotation of a sequence with elementary signals Markup gives information where elementary signals are located in the sequences Complex signals will be build from the elementary signals and operations applied to them Load markup for your sequences in specified XML format or genbank format To skip this step click on the Cancel button To call this dialog again click on the Load markup toolbar button Mapping Sequences You can show loaded sequences by different ways 1 By Positive Negative and Control context menus 259 Unipro UGENE Manual Version 1 20 0 s E Sequences Neg Generate report A Con Export Sequences gt amp Markup Show sequences l Comple myrer 2 By sequence context menu you can show one sequence add sequence to displayed or clear displayed sequences area 4 Positive 4 OAS 2638 m AS S2674 Show one sequence ty S2682 Add to displayed ety 52683 Clear displayed sequences area Jie 53697 TuT m o o a 3 Also by doubleclick on the sequence you can add it to the project Markup Sequences To markup sequences go to the Markup context menu Items 4 Sequences r Positive Wegatrve A Control Marku Complex signals Markup letters Load markup Here you can Markup letters or Load markup Creating Signals To
67. h signal 40586 40643 be Accuracy per residue o 024 4e 01 Bias 1 38025e 02 Conditional e value 1 34634e 01 be Envelope of domain location 40579 40645 HMM region 504 558 be Independent e value 1 34634e 01 be Query sequence Fibronectin 1 2_1 ee Scare 3 513604 H signal 63633 63708 be Accuracy per residue fb235e 01 Bias 4 17314e 03 uMUSCLE UGENE contains graphical ports of the Robert C Edgar s MUSCLE tool for multiple alignment MUSCLE4 is not supported since UGENE version 1 7 2 The package is integrated completely so there is no need in extra files for using it It is possible to run several multiple alignment tasks in parallel check the progress and cancel the running tasks safely The k mer clustering part of the MUSCLE algorithm was optimized for multicore systems by Timur Tleukenov Novosibirsk State Technical University e MUSCLE Aligning e Aligning Profile to Profile with MUSCLE e Aligning Sequences to Profile with MUSCLE MUSCLE Aligning To run the classic MUSCLE use the Align Align with MUSCLE context menu item in the Alignment Editor ER G E A E MO e a Edit d U align with muscLE lily Statistics PN align sequences to profile with MUSCLE View d M Align profile to profile with MUSCLE Advanced eK Align with Kalign 219 Unipro UGENE Manual Version 1 20 0 The dialog contains the list of MUSCLE modes MUSCLE default Large alignment Refine only Align with
68. include the Reference Information if it is available in the assembly file For example e MD5 e Species e URI 154 Unipro UGENE Manual Version 1 20 0 UGENE chrM sam bam sam as chrM WJ File Actions Settings Tools Window Help Beka a okki tJ GR 0 to 16 571 16 571 bp B 079 to 8 143 65 bp a A a ee ee ees ae SES Ap SaaS ASG Ras GSAS ey SSIS eel GIS aa A 8 142C 176 tz 1 Project Assembly Browser Settings The Assembly Browser Settings tab includes Reads Area Consensus Area and Ruler settings Wj File Actions Settings Tools Window Help Goo B A amp oo tiaj amp Reads Area 0 to 16 571 16 571 bp B 079 to 8 143 65 bp Reads highlighting ee oc cre ie correct displaying of this RGRESSIG ET RRP RR Ui SAD LORIE oe ROPE CRETE DUAR APR OTA RT LTR a highlighting 8 1k 8 133C 163 6 140 Scrolling can be optimized by drawing only reads positions without content while scrolling Optimize scrolling t 1 Project V Show pop up hint w Consensus Area Consensus algorithm Difference from reference w Ruler V Show coordinates V Show coverage under cursor 4 To learn more about Reads Area settings refer to the Reads Area Settings chapter To learn more about Consensus see the Consensus Sequence chapter 155 Unipro UGENE Manual Version 1 20 0 To learn more about Ruler see the Browsing and Zooming Assembly chapter
69. is a list of contigs below the Source URL Check the contigs that you want to import to the database You can use the Select All Desel ect All and Invert Selection buttons to manage the selection The Destination URL field specifies the output database file If you check the Import unmapped reads then all unmapped reads in the assembly i e read with the unmapped flag or without CIGAR are imported Note however that they are not vizualized in the current UGENE version To start the import click the mport button in the dialog You can see the progress of the import in the Task View To export a UGENE database file into the SAM format select the Actions Export assembly to SAM format item in the main menu Import ACE File To start working with ACE file you can open it in the Alignment Editor or import it to the UGENE database file To do this open the ace file The following dialog will appear 142 Unipro UGENE Manual Version 1 20 0 uU Select Document Format Open K26 ace as 9 Multiple sequence alignment in the Alignment Editor Short reads assembly in the Assembly Browser If you choose the first option the file will be opened in the Alignment Editor as multiple sequence alignment If you choose the second option the following dialog will appear T Import ACE File C Wwork ugene data samples ACE K26 ace 1 data samples ACE K26 ace ugenedb Select the Source URL and Destination URL and click OK butt
70. likelihood method use fast likelihood method Perform bootstrap the support of the data for each internal branch of the phylogeny can be estimated using non parametric bootstrap Tree searching parameters selection of the tree topology searching algorithm Make initial tree automatically initial tree automatically Type of tree improvement type of tree improvement Set number of random starting tree number of random starting tree Optimize topology the tree topology is optimised in order to maximise the likelihood Optimize branch lengths optimize branch lengths Display tree in new window displays tree in new window Display tree with alignment editor displays tree with alignment editor Synchronize alignment with tree synchronize alignment and tree Save tree to file to save the built tree Press the Build button to run the analysis with the parameters selected and build a consensus tree 140 Unipro UGENE Manual Version 1 20 0 Assembly Browser The UGENE Assembly Browser project started in 2010 was inspired by Illumina iDEA Challenge 2011 and multiple requests from UGENE users The main goal of the Assembly Browser is to let a user visualize and efficiently browse large next generation sequence assemblies Currently supported formats are SAM Sequence Alignment Map and BAM which is a binary version of the SAM format Both formats are produced by SAMtools and described in the following specification SAMtools Su
71. main menu Also when you double click on a read it is Zoomed in and moved to the center of the window By dragging the mouse while holding the left mouse button you can navigate in the Reads Area To navigate long distances in the Reads Area use the Assembly Overview described below Other ways to navigate in the assembly are e Use the horizontal and vertical scroll bars of the Reads Area e Go to a specified position in an assembly To learn about available hotkeys refer to Assembly Browser Hotkeys By default assembly rendering is optimized while scrolling While you are moving across an assembly it shows the assembly in gray color but when you stop it shows the assembly in different colors To disable this option uncheck the Optimize the rendering while scrolling item in the context menu of the Reads Area or Optimize scrolling item on the Assembly Browser Settings tab of the Options Panel Assembly Overview Description The Assembly Overview shows a coverage overview of the assembly The longer the depth of a line in the overview and the deeper the color the more reads are located in this region To open a region of the assembly in the Reads Area click on it in the Assembly Overview On the overview the selected region is displayed either as a gray rectangle a red cross or a red rectangle For example 145 Unipro UGENE Manual Version 1 20 0 If you hold Shift and select a region on the overview the overview is zoomed to the s
72. menu item a Da k a gt ge og 5 a s wog ies sT mo ila moO 1 i e its S 7 5 Ma a a i Cal 7 Parameters i a ae CI Select sequence region Ctrl A Remove ee 5 New annotation CHIN F 2 3 18S oe ee Copy oe ena k te ae The Save Dotplot dialog will appear A dotplot is saved in a file with the dpt extension Later the dotplot can be loaded using the Dotplot Save Load Load context menu item Building Dotplot for Currently Opened Sequence To build a dotplot for currently opened sequences create a multiple view containing these sequences It can be arranged by dragging the corresponding sequence objects the items strated with the s into the same Sequence View Then right click on the created view and select the Analyze Build dotplot item in the context menu Every sequence from the current multiple sequence view can be used to build a dotplot i If you need to compare a sequence with itself you can activate the menu from a single Sequence View Comparing Several Dotplots Dotplots created for the same view are shown at the same view 115 Unipro UGENE Manual Version 1 20 0 If the horizontal and vertical sequences of several dotplots are the same correspondingly it is possible to lock all zooming and navigating operations for these dotplots Press the Multiple view synchronization lock button on the left 3
73. multiple sequence alignment object in the database after the import is done Create a subfolder for each document if this option is checked for each document or object uploaded to the database a new folder is created having the same name as the file and the data are placed in the folder Otherwise the data are imported into the Destination folder Database in the Project The database in UGENE Project View looks like as a tree with folders and objects my_database 4 gt 1CRN prt 4 3d 1CRN hy s LCRN chain 1 sequence Y a 1CRN chain 1 annotation gt 2QS prt gt gt ABIF a gt ACE 4 gt BLO60C3 ace A s Contig _ref as Contigl A s Contigl_ref as Contig gt W6 ace Assembly CLUSTALW EMBL oao Genbank MSF Recycle bin You can add a new folder to the database tree To do that use the Add gt Aadd folder database context menu item To add a subfolder to some existing folder use the Add gt Add folder folder context menu item To delete an object or a folder press the Delete button or drag n drop it to the Recycle bin In this version of UGENE objects in the database are read only Nevertheless there is a workaround to edit them First you need export the objects to files on your computer using the Export Import object context menu Then you can change that files locally upload them to database and finally delete the originals If new data are added to the database by another user or rem
74. nucleotides To add a 5 overhang to the reverse strand in addition to the described steps select the Reverse complement item in the same group box Similarly to add a 3 overhang check the Include Right Overhang check box input the required overhang and select either the direct or the reverse complement strand On the Output tab of the dialog you can optionally modify the annotations output settings Finally press the OK button to create the fragment The fragment will be saved as an annotation 194 Unipro UGENE Manual Version 1 20 0 Constructing Molecule To construct a new molecule from fragments select the Tools Cloning Construct Molecule item in the main menu If a Sequence View window is active you can also select either the Actions Cloning Construct Molecule item in the main menu or the Clonin g Construct Molecule item in the context menu The Construct Molecule dialog appears T Construct Molecule New molecule contents Fragment Inverted Annotate fragments in new molecule E Force blunt and omit all overhangs E Make crcular Available Fragments Fragments of the New Molecule Changing Fragments Order in the New Molecule Removing Fragment from the New Molecule Editing Fragment Overhangs Reverse Complement a Fragment Other Constuction Options Output Available Fragments All the fragments available in the current project are shown in the Available fragments list You can automatically create
75. of several genes involved in the cellular response to DNA damage or inhibition of DNA replication 208 Lrp MALT MARA MELR MEtJ MetR1 MLC MODE NAC NAGC_new2 NANR NARL2 NARL NARP NIRC OmpC OxyR PHOB PHOP PurR RcsB_1 RcsB_2 Rob2 ROB soxS TORR TRPR TyrR Building SITECON Model Unipro UGENE Manual Version 1 20 0 Leucine responsive regulatory protein Maltose regulator Multiple antibiotic resistance Melibiose regulator MetJ represses the expression of genes involved in biosynthesis and transport of methionine MetR participates in controlling several genes involved in methionine biosynthesis Weissbach91 and a gene involved in protection against nitric oxide DgsA better known as Mic makes large colonies is a transcriptional dual regulator that controls the expression of a number of genes encoding enzymes of the Escherichia coli phosphotransferase PTS and phosphoenolpyruvate PEP systems Molybdate responsive transcription factor Nitrogen assimilation control N acetylglucosamine N acetyl neuraminic acid regulator Nitrate nitrite response regulator NarL Nitrate nitrite response regulator NarL Nitrate nitrite response regulator NarP NirC is a nitrite transporter which is a member of the FNT family of formate and nitrite transporters OmpC is a member of the GMP family Oxidative stress regulator PhoB is a dual transcription regulator that activates expression of t
76. output Short reads Path Max diff n 0 Max gap opens 0 1 O menopo Gn 00 2 exten ca Seed length H 2 Best hits R 30 4 E Colorspace c E Long scaled gap penalty for long deletions 1 E Nonterative mode N 230 Unipro UGENE Manual Version 1 20 0 There are the following parameters Reference sequence DNA sequence to align short reads to This parameter is required Result file name file in SAM format to write the result of the alignment into This parameter is required Library single end or paired end reads Prebuilt index check this box to use an index file instead of a source reference sequence Also you can build it manually SAM output always save the output file in the SAM format the option is disabled for BWA Short reads each added short read is a small DNA sequence file At least one read should be added You can also configure other parameters They are the same as in the original BWA you can read detailed description of the parameters on the BWA manual page Select one of the following parameters that correspond to the n option in the original BWA Max diff n maximum edit distance An integer value should be input Missing prob n the fraction of missing alignments given 2 uniform base error rate A float value is used Seed length I take the subsequence of the specified length as seed If the specified length is larger than the query sequence seed
77. reads method parameter to Bowtie 2 The dialog looks as follows M Build Index Align short reads method Reference sequence Index file name There are the following parameters Reference sequence DNA sequence to which short reads would be aligned to This parameter is required Index file name a file to save the created index to This parameter is required BWA BWA is a fast light weighted tool that aligns relatively short reads to a reference sequence Click this link to open BWA homepage BWA is embedded as an external tool into UGENE 229 Unipro UGENE Manual Version 1 20 0 Open Tools DNA assembly submenu of the main menu Tools Window Help DNA assembly Align short reads Test runner Build index 45 SITECON i 32 Convert UGENE Assembly data base to SAM format Select the Align short reads item to align short reads to a DNA sequence using BWA Or select the Build index item to build an index for a DNA sequence which can be used to optimize aligning of short reads e Aligning Short Reads with BWA e Building Index for BWA Aligning Short Reads with BWA When you select the Tools DNA Assembly Align short reads item in the main menu the Align Short Reads dialog appears Set value of the Align short reads method parameter to BWA The dialog looks as follows I Align Sequencing Reads Alignment method Reference sequence Result file name Library F Prebuilt index SAM
78. the view of the Advanced options tab is the following 183 Unipro UGENE Manual Version 1 20 0 A Request to Local BLAST Database General options Advanced options Extension options Threshold 12 00 Filters Masks E Low complexity filter E Mask for lookup table only E Human repeats filter Mask lower case letters As you can see there is no Match scores option but there are Threshold Matrix Composition based statistics and Service options Threshold threshold for extending hits Matrix key element in evaluating the quality of a pair wise sequence alignment is the substitution matrix which assigns a score for aligning any possible pair of residues Service blastp service which needs to be performed plain psi or phi Composition based statistics composition based statistics When the tblastx search is selected in the general options the view of the Advanced options tab is the following 184 Unipro UGENE Manual Version 1 20 0 A Request to Local BLAST Database Advanced options w Word size 3 Threshold 13 00 Filters Masks E Low complexity filter E Mask for lookup table only E Human repeats filter Mask lower case letters Search The following extension options are available A Request to Local BLAST Database X dropoff value in bits For gapped alignment For ungapped extensions For final gapped alignment Multiple Hits Window Size Perform gapped alig
79. time to calculate the overview and the well covered regions To see the reads either select a region from the list or zoom in for example by clicking the link above the well covered regions or by rotating the mouse wheel You can also use the hotkeys Tips about hotkeys are shown under the list of well covered regions To learn about available hotkeys refer to Assembly Browser Hotkeys Assembly Browser Window Components An Assembly Browser window consists of Assembly Overview By default shows the whole assembly overview Can be resized to provide an overview of an assembly part Reference Area Shows the reference sequence Consensus Area Shows the consensus sequence Ruler Shows the coordinates in the Reads Area Reads Area Displays the reads Coverage Graph 144 Unipro UGENE Manual Version 1 20 0 Shows the coverage of the Reads Area See the example below Assembly Overview ET Reference Area 0 to 10 O00 10 000 bp 2 646 to 2 732 87 bp Consensus Area FR RSS Pano aerials ania Saran dane mene rena ana ie fanaa ana 3 Ruler iaman 2 F32 C 20 Coverage Graph gt bees Ill lt 4 Reads Area Description The Reads Area provides a visualization of reads of an assembly part To zoom in or zoom out rotate the mouse wheel To perform zooming you can also use the Zoom In and Zoom Out buttons on the toolbar or the Actions Zoom In and Actions Zoom Out item s in the
80. to compute a distance matrix The following values are available for a nucleotide multiple sequence alignment e F84 e Kimura e Jukes Cantor e LogDet The following models are available for a protein alignment e Jones Taylor Thornton e Henikoff Tillier PMB e Dayhoff PAM e Kimura Gamma distributed rates across sites specifies to take into account unequal rates of change at different sites It is assumed that the distribution of the rates follows the Gamma distribution Coefficient of variation of substitution rate among sites becomes available if the Gamma distributed rates across sites parameter is checked Specifies the coefficient of the distribution of the rates Transition transversion ratio expected ratio of transitions to transversions To enable bootstrapping check the Bootstrapping and Consensus Trees group check box The following parameters are available Number of replicates number of replicate date sets Seed random number seed By default it is generated automatically You can manually change this value in order to make results of different runs of a tree building reproducible The should must be an integer greater than zero and less than 32767 and which is of the form 4n 1 that is it leaves a remainder of 1 when divided by 4 Any odd number can also be used but may result in a random number sequence that repeats itself after less than the full one billion numbers Usually this is not a problem
81. to query The algorithm applied in RT PCR primer design first searches for all available primers in a given sequence Then it filters the detected pairs to make sure that they satisfy the selected configuration This option allows to set the maximum number of pairs for the initial search query Larger number will result in increased sensitivity but also in a longer running time Default value is 1000 Important using the RT PCR primer design tab will reset the values set in the Ex cuded regions and Targets of the Main configuration tab Additionally if the Exon range option is set the defined sequence region will be ignored 252 Unipro UGENE Manual Version 1 20 0 Spliced Alignment mRNA to genomic UGENE allows to align spliced mRNA cDNA sequence to genomic sequences The default underlying algorithm which is used for the alignment is an external tool called Spidey Before running the alignment make sure that Spidey is available and validated in the list of External Tools To perform the alignment of a mRNA sequence to a genomic sequence open the the genomic sequence in the Sequence View Next activate context menu item Align gt Align to sequence to MRNA F L L R R 85 L tr Go to position Ctrl G M e L n E r TC Select sequence region Ctrl A ATGGTGCATCTGACTCCTGAGGAGAAGTCTOC S A New annotation Ctrl N 55 60 65 70 75 80 TACCACGTAGACTGAGGACTCCTCTTCAGACE Copy p Select Add Ana
82. values of qualifiers recalculates regions in qualifiers when sequence is modified Position to insert the sequence position where to insert the subsequence Save to new file the result sequence can be saves to a new file instead of modifying the current file You must select the Document location FASTA and Genbank file formats are available when you do not include annotations to the result file If you check the Merge annotations to this file item the annotations will also be saved to the result file Genbank file format is only available in this case In case a subsequence has been selected the Replace subsequence is available from the context menu or by the Ctr R shortcut The dialog opened in this case is similar to the dialog described above except it already contains the sequence to be edited an doesn t allow to input the start position Also it is possible to remove selected subsequence from a sequence When you select corresponding item in the context menu or in the Acti ons menu the Remove subsequence dialog appears 62 Unipro UGENE Manual Version 1 20 0 A Remove Subsequence Region to remove 37 74 Annotations region resolving mode Crop corresponding annotation Remove corresponding annotation Recalculate values of qualifiers Save to new file Merge annotations to this file Document location jples Genbank murine_new fa Document format FASTA Description of the parameters Region t
83. wD DM 40 5 86 70 SO g 100 110 120 130 140 1580 160 170 180 190 200 a 2 0 00 Geeceacooo a G AT CAAAG ACA G il Wath 102 ne en ne en ee ee 98 99 100 101 102 103 1 ds ie 6 107 108 H2 113 114 15 116 117 118 19 I2 GGA TC AA AGACAG gt 901 07 copy sequence ENA fi cls amp BQ o 110 2030 80 90 100 110 120 130 140 150 160 170 180 190 200 ao G eoqgerceoeeoooeeeo ee eee ee0 eG i AOTEA 14 44 1R Wale cee o Se Geeks T Sela a Ga aad as OE Yalue tA No actiwe tasks g DNA RNA Graphs Package The DNA RNA Graphs Package draws contextual graphs for sequences The DNA RNA Graphs Package is available for the Standard DNA and Standard RNA alphabets Open a sequence in the Sequence View and click the Graphs icon on the toolbar The popup menu appears DNA Flexibility i 120k sok 4 GC Content 5 AG Content 180k 190k 1 6C Frame Plot GC Deviation G C G C 2 AT Deviation 4A 1 4A4T L O A R R Karlin Signature Difference i R p F F Informational Entropy p T z n A R CCTACACCCCACAACAG AGCCCATCAGACTAACAGCGt oe ee ee ow RS Sa aa ee T E To see a graph select the corresponding graph item in the popup menu A new area with the graph appears right above the Sequence zoom 120k 130k 140k 120k 130k 140k K 5 I R L tT LO x H H e T N S dL view 102 Unipro UGENE Manual Version 1 20 0 H murine NC_001363 ra O amp S cel T ok GC Frame
84. zoom view when the sequence is not zoomed use the Zoom to Whole Sequence bution Creating New Ruler You can create any number of additional rulers by clicking the Ruler Create new ruler context menu item 53 Unipro UGENE Manual Version 1 20 0 Go to position Ctrl G i L E P Select sequence region CtriA a i New annotation Ctri N TTTTTG CC AAC G Rename item 1245 1250 1255 LAAAAACTTTGGTTGC Copy Paste E E F G y select a s i cece i s p Add peeassssesssesisessresirssiresiresirearenrnnrn Align Cloning Export Edit Remove Rulers Create new ruler Statistics Show Main Ruler Show Custom Rulers The following dialog will appear Create New Ruler Ruler name New ruler Ruler start Ruler color Sample Text The new ruler will be shown right above the default one T E a z T 5 New ruler with a custom offset Y Wf F T Y F A FE 5 L We L F 5 E I R F L P T S E E P T H L D L L OF I i 2 L WN STATTACGOGTTTTTACCTACCCACGOGAAAAGC CAAC CAAC C TCOATCTCOCTTOTACGATCTOITTCOTCOTAAA 71 d Cc Lm ano ad 10 40 97 77 Wd C WS SF ST Dd TC 0 Ar A dd ic qo En OC ad cc o On O27 Ca T Selecting Amino Translation The default value for the genetic code is read by UGENE from the sequence file when it is available You can also select the genetic code for the sequence using the Amino translation menu button on the sequence toolbar A All analysis routines l
85. 13525 299
86. 150 200 250 J300 350 400 450 500 550504 pg 4 it fasta_example fasta b Phaneroptera_falcata s Isophya_altaica_EF540820 s Bicolorana_bicolor_EF5 40830 s Roeseliana_roeseli Te de 40 40 96 gol 46 Kel a6 46 40 Open view A L Add to view TCGAGCCGAATTAGGTC AAC ee eae 4 16 18 20 22 24 26 28 30 33 oa ca anaes AGC TCGGCTTAATCCAGTTG Ss ane Lock document for editing R 7 N P r i s Zychia_baranovi He s Tettigonia_viridissima s Conocephalus_ discolor _ Import s Conocephalus_sp Edit fasta_exarmple_fasta Remove B Save selected documents No active tasks gy The Export Selected Sequences dialog will appear Export Selected Sequences Export to file C work ugene data samples FASTA human_T1_region_new fa File format to use Export with annotations Add document to the project cename human_T1_region Converton options Save direct strand Save complement strand O Save both strands E Translate to amino alphabet Save all amino frames Use custom translation table 1 The Standard Genetic Code Merge options Save as separate sequences Merge sequences Add gap symbols between sequences Here you can select the location of the result file and a sequence file format You can choose to add newly created document to the current project and use custom sequence name To do it check the corresponding checkboxes Use the Conversion options to choose a strand for saving sequence s Als
87. 2008 Vityaev Kovalerchuk 2004 Kovalerchuk Vityaev 2000 The approach was used in Discovery system which has been successfully applied for solution some particular problems in the fields of psychophysics cancer diagnostics and securities rates prediction The heart of the system is semantic probabilistic inference Vityaev 2006 257 Unipro UGENE Manual Version 1 20 0 The idea of new knowledge discovery is to sequentially increase accuracy of hypotheses so that on each step the hypotheses have the higher probability and definition level Also the level of significance of the results is tested by statistical criterions Discovery system implements semantic probabilistic inference with knowledge discovery as a set of probability laws the strongest probability laws and maximally specific laws ExpertDiscovery is an adaptation of the Discovery system which is configured to knowledge discovery in sets of nucleotide sequences according to semantic probabilistic inference as complex signals with specified parameters ExpertDiscovery plugin in UGENE has the following advantages 1 Crossplatforming 2 The unite system a Many algorithms within the bounds of one project apparently give more possibilities than many different individual narrow applications Such an approach simplifies user s work that is needed is to launch UGENE which gives the access to the wide range of the algorithms instead of launching different unrelated programs
88. 3 799 30k 60k fa 167 3 43 799 20k 60k fa 167 Multiple view synchro I r 1 h tal S rto ON ba aauanbas z P oh F aauenbas zgzrT0 ON FOK eos 2 ee le NC_014267 sequence min length 11 identity 100 NC_014267 sequence min length 15 identity 100 116 Unipro UGENE Manual Version 1 20 0 Alignment Editor e Overview e Alignment Editor Features Alignment Editor Components Navigation Coloring Schemes e Creating Custom Color Scheme Highlighting Alignment Zooming and Fonts Searching for Pattern Consensus e Export Consensus e Alignment Overview e Working with Alignment e Undo Redo Framework e Selecting Subalignment e Moving Subalignment e Editing Alignment e Removing Selection Filling Selection with Gaps Replacing with Reverse Complement Replacing with Reverse Replacing with Complement Removing Columns of Gaps e Removing All Gaps Saving Alignment Aligning Sequences Aligning Sequence to this Alignment Pairwise Alignment Working with Sequences List e Adding New Sequences e Copying Sequences Renaming Sequences Sorting Sequences Shifting Sequences e Collapsing Rows e Exporting in Alignment e Extracting Selected as MSA e Exporting Sequence from Alignment e Exporting Alignment as Image e Statistics e Distance Matrix e Grid Profile e Advanced Functions e Building HMM Profile e Building Phylogenetic Tree e PHYLIP Neighbor Joining e MrBayes e PhyML Maximum Likelihood Overview
89. 3258 If you work with file with many sequences the button closes circular views if some circular views are opened and if all circullar views are closed it opens all of them Also you can mark sequences as circular in UGENE by the Mark as circular sequence context menu item When the sequences are marked as Circular the Circular View is automatically opened for them in all opened Sequence View windows The Restriction Sites Map will appear automatically To show restriction sites the Show Restriction Sites menu should be checked To hide the map click on the following button 85 Unipro UGENE Manual Version 1 20 0 Actions Settings Q File a OS a A The Circular Viewer is opened automatically when the Sequence View is opened for a plasmid The inner circle represents the sequence clockwise and the scale marks show the corresponding sequence positions The sequence annotations are represented as curved colored regions at the outer side of the circle The Circular Viewer helps to navigate within the sequence You can select an annotation on the circular view and the annotation will also be focused and highlighted in all Sequence View areas Sequence overview Sequence zoom view Sequence details view and Annotations editor You can also select a sequence region EBY_rev_primer pBR322_ origin is T _promoter fa pCAG_F_pafifer bGlob_int SV40pro_F_primer SV40_origin oV40_promoter x Tale p p
90. 50 Penalty unpaired U Skip seeds threshold c 10000 Score threshold T Drop chain threshold D 0 50 Rounds of mate rescues m E Skip mate rescue S E Skip pairing P NOTE bwa mem accepts reads only in FASTA or FASTQ format Reads should be compiled into a single file for each mate end There are the following parameters Reference sequence DNA sequence to align short reads to This parameter is required Result file name file in SAM format to write the result of the alignment into This parameter is required Prebuilt index check this box to use an index file instead of a source reference sequence Also you can build it manually SAM output always save the output file in the SAM format the option is disabled for BWA Short reads each added short read is a small DNA sequence file At least one read should be added You can also configure other parameters Index algorithm a algorithm for constructing BWA index 236 Unipro UGENE Manual Version 1 20 0 It implements three different algorithms e is designed for short reads up to 200bp with low error rate lt 3 It does gapped global alignment w r t reads supports paired end reads and is one of the fastest short read alignment algorithms to date while also visiting suboptimal hits e bwtsw is designed for long reads with more errors It performs heuristic Smith Waterman like alignment to find high scoring local hits Al
91. 63 features murine gb 4 cos 0 4 E CDS CDS 1042 2658 E CDS CDS join 2970 3413 E CDS CDS 3875 4999 E CDS CDS 5048 5203 w comment 0 1 a w misc_feature 0 2 E misc_feature Misc Feature 2 590 E misc_feature Misc Feature 5245 5833 w source 0 1 Statistics wv Common Statistics gt Characters Occurrence w Dinucleotides Length 589 GC Content 52 12 Molar Weight 181766 49Da Molar Ext Coef 6352900 I mol Melting TM 85 13C nmole OD20 0 16 pg OD2 lt 0 28 61 BES PesEBS 37 37 32 AHARPABARIAKRARAS BAG To copy the statistical information about a sequence select it on the Options Panel and choose the copy item in the context menu or use the Ctrl C shortcut Manipulating Sequence Going To Position Toggling Views Exporting Sequence Image Zooming Sequence Creating New Ruler Selecting Amino Translation Showing and Hiding Translations Selecting Sequence Copying Sequence Search in Sequence e Load Patterns from File e Search Algorithm e Search in e Other Settings e Annotations Settings Editing Sequence Exporting Selected Sequence Region 51 Unipro UGENE Manual Version 1 20 0 e Exporting Sequence of Selected Annotations e Locking and Synchronize Ranges of Several Sequences e Multiple Sequence Opening Going To Position To go to a position use the global actions toolbar Or use the Go to position context menu or the Actions main menu item C H N E M K ray F Go
92. 875 sequence Activate view Space 4dd bookmark z f Fa Rename bookmark F Remove bookmark Del r l E For every persistent view UGENE automatically saves the state of the view in the Auto saved bookmark when the view is closed Now by activating bookmarks you can restore the original view state For example for the Sequence View bookmarks you can store a visual position and zoom scale for the sequence region OP a NT_025441 features O s NT_025441 sequence a NT_025975 Features s NT_025975 sequence a NT_078122 features GP s NT_O76122 sequence W hs_chrY NT_011875 sequence i J 10 15 a 23 CITAAGTAAGCTTATCTTAACTTAGC I E E L I D F E N 585 Y F Q I 5 N M R I 5 N FE R Nane NT_011875 features hs_chr gbk gz 3 Auto sawed Activate view ET Add bookmark i Rename bookmark Space F2 Del Use the F2 keyboard shortcut to rename a bookmark To remove a bookmark press the Delete key UGENE has limited set of built in Object Views Extensions modules or plugins can be used to adjust the existing views or to add new views to the tool Exporting Project All the opened documents and bookmarks along with the corresponding views states can be saved within a project file To do so select File Export Project It will invoke the Export project dialog where you can select the destination folder and the project file name 33 Unipro UGENE Manual Version 1 20 0
93. Annotations Editor e Open the context menu e Choose the Fetch sequences from remote database gt Fetch sequences by id from blast result item or Fetch sequences from remote database gt Fetch sequences by accession from blast result item The following dialog will appear A Get Sequences by ID The sequences from selected BLAST results will be downloaded fram NCBI Genbank by their GI identifier Save to directory Add to project Select an output path in the dialog and click the OK button BLAST BLAST The Basic Local Alignment Search Tool BLAST finds regions of local similarity between sequences The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches BLAST can be used to infer functional and 179 Unipro UGENE Manual Version 1 20 0 evolutionary relationships between sequences as well as help identify members of gene families BLAST is a new version of the BLAST package from the NCBI From UGENE you can use the following tools of the old BLAST package e blastall the old program developed and distributed by the NCBI for running BLAST searches e formatdb formats protein or nucleotide source databases before these databases can be searched by blastall And the following tools of the new BLAST package e blastn searches a nucleotide database using a nucleotide query e blastp searches a protein database usi
94. Application Settings General File Format Resources Network sequence annotations File Format Logging Alignment Color Scheme External Tools Genome Aligner OpenCl Workflow Designer Create annotations for case switchings Don t use case annotations The Sequence Annotations settings allows to use upper lower case annotations during the file reading process Format options 1 Don t use case annotations default mode usual sequence reading and writing 2 Use lower case annotation sequences are read and annotations with names lower_case are added When these sequences are written to file then the case becomes like original the file case the case is saved 3 Use upper case annotation there is a similar behavior but with upper_case annotations Directories 41 Unipro UGENE Manual Version 1 20 0 m Application Settings General Directories Resources Network File Format Directories Logging Alignment Color Sch Path to downloaded data Path for temporary files External Tools Genome Aligner File storage Workflow Designer OpenCL Cleanup storage The following settings are available on the tab Path to downloaded data specifies the path where files downloaded from the remote databases will be stored Path for temporary files the path where will be stored temporary files File storage the path where will be stored UGENE files Logging 42 Unipro UGENE Manual
95. C_001363 W murine s NC_001363 2 vy 1CF7 PDB 2 Bookmarks Log view UGENE Window Components This chapter describes UGENE main window components Project View Task View Log View and the Notifications popup window e Welcome Page Configure the annotation type Show annotations of this type Show on translation Show value of qualifier protein_id locus_tag gene function p Notifications Active task No active tasks 9g E Unipro UGENE Manual Version 1 20 0 e Project View e Task View e Log View e Notifications Welcome Page The Welcome Page is the first page that will appear when UGENE has been launched From the Welcome Page you can open files create sequence create workflow open the Quick Start Guide and open recent files directly T UGENE Start Page U File Actions Settings Tools Window Help cx Welcome to UGENE Recent files 538117317_misc3_copyl aln OM 538117317_misc3 nwk 538117317_misc3 aln Open File s Create Sequence i mk P19121 gb P49822 gb Recent projects No opened projects yet Create Workflow Quick Start Guide To return to the Welcome Page go to the Window gt Start Page main menu item Project View The Project View shows documents and bookmarks of the current project The documents are files added to the project And the bookmarks are visual view states of the documents Read Using Bookmarks to learn more about bookmarks To show hide the Project Vi
96. EF540828 Hetrodes_pupus_EFS40832 Isophya_altaica_EF540820 Metrioptera_japonica_EF540831 Montana _montana Phaneroptera_falcata Podisma_sapporensis Roeseliana_roeseli Tettigonia_viridissima To update the collapsed groups click on the corresponding main toolbar button Exporting in Alignment e Extracting Selected as MSA e Exporting Sequence from Alignment e Exporting Alignment as Image Extracting Selected as MSA It is possible to extract a subalignment and save it as new multiple sequence alignment MSA Select a subalignment and choose the Export Save subalignment item in the Actions main menu or in the context menu The following dialog appears 131 Unipro UGENE Manual Version 1 20 0 WEEE Extract Selected as MSA From Selected sequences Phaneroptera_falcata Isophya_altaica_EF540820 Bicolorana_bicolor_EF540830 Montana_montana Metrioptera_ japonica _EF540831 Gampsocleis_sedakovil_EF 540828 Deracantha_deracantoides_EF540 Clear selection File name ita samples CLUSTALW COI_subalign 1 aln File format to use CLUSTALWY Add to project Specify the name and format of the new MSA file in the File name and File format to use fields The currently selected region is extracted by default when you press the Extract button You can change the columns to be extracted using the From and to fields And change the rows to be extracted by checking unchecking required sequences in the Selected sequences list
97. ER Pi 589 bp 5590 2 Aiae aa INC 00136 E Objects with annotations Column with L L gP NC 001363 features murine gb note qualifier W cos 0 4 values O cbs Groups 1042 2658 O cbs join 2970 3413 3412 3873 Predicted By GeneMark artific O cbs 3675 4999 0 cps Annotation 5048 5203 Predicted by GeneMark G misc_feature 0 yo misc_feature 2 590 5 terminal repeat note 5 terminal repeat O misc_feature 5245 5833 F terminal repeat th y source 0 1 Qualifiers name and value LIL There are usually several objects with annotations in the Annotations editor A special Auto annotations object is always presented for each sequnce opened It contains annotations automatically calculated for the sequence see below for details An object contains groups of annotations used by UGENE for logical organization of the annotations An annotation must always belongs to some group For documents created not by UGENE annotations are grouped by their names For annotations created in UGENE it is possible to use arbitrary group names Groups can contain both annotations and other groups The numbers in the brackets after a group name in the Annotations editor are the count of subgroups and annotations in the current group A single annotation is allowed to be presented in several groups simultaneously An annotation is physically removed from the document when it does not belong to any group e db xref Qualif
98. GENE murine s NC_001363 4 File Actions Settings Tools Window Help Boek A f amp fo Ee ka g NC_001363 dna ok 4k cs CCAC CGOGGAGGOGTAAGCTGOGCCAGCAACTTATCTGOTGOGTCTOTCCGATTOTCTAGTOT a a aaa y SAHA T i641 645 650 655 665 100 1k 100 2k CE TTGTCAGATTCACCAAACTTGAAATGAAGCGGAAAAAATGCTAAGCGGGCAGCGCCAGAGAG r eee eee 12 4 6 6 10 12 14 16 18 20 2 24 2 26 30 2 M 5 5 UA 42 H 4 B 80 a MS i _ Type cy Auto annotations hurma No active tasks 9g E You can change the focus by clicking on the corresponding sequence area All sequences that are not in focus have the sequence name and icon disabled The bottom area of the Sequence View is the Annotations Editor It contains a tree like structure of all annotations available for all sequences shown in the Sequence View and can be used to perform various actions on annotations create a new annotation modify the existing one group sort etc Global Actions Riv Wa The global action toolbar provides possibility to go to the specified position in all sequences at the same time Also it allows to lock or adjust ranges of sequences in the same Sequence View See this paragraph for details Sequence Toolbars A brief description of the sequence toolbars buttons is shown on the picture below 48 Unipro UGENE Manual Version 1 20 0 NC_001363 dna Toggle annotation density graph Ok B LE TT TT SY FEET PP L Sh aA A
99. ITGATATIC AGATACAAGGAGAAAATATGCCAGTAAGAAAATGCATITITCAAGATTIAAATTCGGCATT TGTTACTIAATAGCATTIGICATATICCAATITITCATATGIAGTAAATICATITCAAAT CA SENSGOO000232606 ENSTOO000413525 cds KNOWN lincRNA 2ENSG00000232606 ENST00000413525 ENSEOO001765550 exon KNOWN lincRNA AGCTTCACATGTGAGATASATGCACTCAAAGATTCCTCACAAGTAGCTCITTGGAGCTIC AGAT GT GAAATGGATCATICCTCAATCTGIAATAGACCCTICTIGIGAAGCTCTICAATCA AACCAGAGAATTCAAG gt ENSGO0000232606 ENSTOO000413525 ENSEOO0017095330 exon KNOWN lincRNA AGTTTCCAACACCTAAGAGTGGOTATTTGGOCAAAT GOT GOGCCAAAGGAATAAAGAAGGCA TGOCASAACTCTTGACAGASAGACATTCAGASATTIGATTITGATATCAGATACAAGGAGAAAA TATGCCAGTAAGASAAATGOCATITITCAAGATTAAATTCOGCATTITGITACTTAATAGCAT TIGTCATATTCCAATTTTITCATATGOTAGTAAATTOCATTTCAAAT CA PENSGOO0002326 Copy Ctrl C GTGAAATIGAGGGSE AGGAACCTGAGGTISA AAGGCTGICAATCS AGACAAGCTGIAL TeacnacectTTace LU Open selection in UGENE CTCTCTIATTIGAATI a i GAAAAAACATTAGI nspect elemen AAGAATTGATATATTITATAAAGTAATGATCCTCATAGTTACATCTIATTTIAGGGATTAT GITTGTAGATCATGTAATGAGTTITAATAAGTITGITICCAGTASAGCAGTAAGACAGAG TIACCTCIGGTAAGGAAAAAATAAAGAAGATCTATICIGASATIAGITTITGIGICACAT TITTAATIGATITGGIGCCACATTITCCAGITGGACATITCTAGGITAATITITITITATT ATACTIGAAGTITIAGGGTACACGTGCACAACGIGCAGGITACATATGTATACATGTGCC ATGITGGIGTGCTGCACCCATTAACTCOTCATTTAACATIAGGIATATCICICIAGIITA ATTTTTAAGAAACTCTAGAATTTTCAATGGGCTATTTIAGITTGGAGAAGCAGTGAATCCT CACACCAAACCAACTAAT CAAAACTOTOCTTTACAATTTATOCTOAAATATOCACTATAET Print Search Google for gt ENS G00000232606 ENS 7000004
100. Indude files filter fa fasta Exdude files filter pal Type of file s protein nudeotide Output settings Select the path to save the database into Base name for BLAST files Tite for database file Format Here you must select the input files If all the files you want to use are located in one directory you can simply select the directory with the files By default only the files are taken into account with fa and fasta extensions You can change this by specifying either Include files filter or Exclude files filter You can choose either protein or nucleotide type of the files Then you must select the path to save the database file and specify a Base name for BLAST files and a Title for database file Making Request to Database To make a request to a local BLAST database do the following e lf you re using BLAST open Tools BLAST BLAST Search e If you re using BLAST open Open Tools BLAST BLAST Search If there is a Sequence opened you can also initiate the request to a local BLAST database from the Sequence View e If you re using BLAST select the Analyze Query with BLAST item in the context menu or in the Actions main menu e If you re using BLAST select the Analyze Query with BLAST item in the context menu or in the Actions main menu The Request to local BLAST database dialog will appear 181 Unipro UGENE Manual Version 1 20 0 4 Request to Local BLAST Database General options Ad
101. Molar weight is the sum of the atomic masses of the constituent atoms for 1 mole of oligonucleotide e Molar ext coefficient the molar extinction coefficient is a physical constant that is unique for each sequence and describes the amount of absorbance at 260nm A of 1 mole L DNA solution measured in 1 cm path length cuvette e Melting TM melting temperature is the temperature at which an oligonucleotide duplex is 50 in single stranded form and 50 in double stranded form e nmole OD the amount of oligonucleotide in nanomoles that when dissolved in 1 mL volume results in 1 unit of absorbance at 260 nm with a standard 1 cm path length cuvette e g OD 6 the amount of oligonucleotide in micrograms that when dissolved in 1 mL volume results in 1 unit of absorbance at 260 nm with a standard 1 cm path length cuvette e Characters occurrence e Dinucleotides occurrence for sequences with the standard DNA and RNA alphabets NC_001363 dna Ow gt Gs w amp 24 Vv 5k 58332 1 500 1k 1 5k 2k 2 5k k 3 5k 4k 4 5k 4 1 500 1k 1 5k 2k 2 5k BK 3 5k 4k 4 5k 5k 53833 589 bp 590 4 p v z M TAGCAGCCATTGCCGTACTGACAAAGGATGCAGGCAAGCTAACCATGGGACAGCCAC ce Tc cc cc cc cc cc rT 2974 2980 2985 2990 2995 3k 3005 3010 3015 3020 3025 3030 ATCGTCGGTAACGGCATGACTGTTTCCTACGTCCGTTCGATTGGTACCCTGTCGGTG M L M L L L L 4 Name a Type Value g Auto annotations murine gb NC_001363 a g NC_0013
102. O repeat unit join 6149028 6149029 6149030 6149031 6149032 6149033 6149034 6149035 6149036 6149037 6149038 6149039 O repeat unit join 4607596 4607598 4607599 4607601 4607602 4607604 4607605 4607607 4607608 4607610 4607611 4607613 M repeat unit _ioin 8049749 8049752 8049753 8049756 8049757 8049760 8049761 pita it ADAG 76S 8049768 8049769 pode772 an Mi fl The tandem repeats annotations are located side by side Restriction Analysis 190 Unipro UGENE Manual Version 1 20 0 From this chapter you can learn how to search for restriction sites on a DNA sequence The restriction sites found are stored as automatic annotations This means that if the automatic annotations highlighting is enabled then the restiction sites are searched and highlighted for each nucleotide sequence opened Refer Automatic Annotations Highlighting to learn more Open a DNA sequence in and click the following button on the Sequence View toolbar k Alternatively select either the Actions Analyze Find restriction sites item in the main menu or the Analyze Find restriction sites item in the context menu The Find restriction sites dialog appears 9 Find Restriction Sites Filter by name Name Accession Sequence Organizm Details gt A 0 264 Aaal Axyl gt BG 917 Bacl BvuBl gt C 1 193 Cacl Cvnl gt D1 31 Daal DsplI gt E 1 325 Eacl EspHK30I gt FO 63 F Cphl F TevIV ac
103. PBABE_3_primer AmMpR promoter M13_forward20_ primer pGEX_3 primer 4 m 2k 2 9k 3k 3 3k 4076 This will also affect the Sequence View You can select a sequence region with Ctrl and the selection will be inverted Note that the circular view is zoomed automatically when the Circular Viewer area is resized 86 Unipro UGENE Manual Version 1 20 0 EBV rev_primer T _ promoter E pCAG_F primer ei Resizing the a ale ai circular view area 4 is resizing the sequence circular view sV40_ promoter ee sV40pro_F primer SV40_ origin M13_forward20_ primer 00 1k 1 5k 2k 2 3k 3k 3 3k 4 076 So you can adjust it to an appropriate size It is possible to rotate the circular view using the mouse wheel Also it is possible to shift the start point of a circular molecule by Edit sequence gt Set new sequence origin context menu item Use the Export Save circular view as image context menu or the Actions main menu item to save the image of the circular view EBV_rev_primer PBR322 origin T7 promoter Y f pCAG_F_primer gt I bGlob int Goto position Cirl G Select sequence region Ctrl A A New annotation Ctrl M Copy j 7 AmpR_promoter Select e po j Add Analyze Align Cloning d Export fe Save circular view as image Edit sequence Export selected sequence region The Export Image dialog will appear 87 Unipro UGENE Manual Version 1 20 0 l Export Image
104. Plot 1 5833 Window 30 Step 10 h 5833 Window 100 FA i COT AGG Auto annotations murine gb NC_001363 eh cy NC_001363 features murine gb 4 Each point on a graph is calculated for a window of a specified size The window is moved along the sequence by a step See Graph Settings for instructions on how to modify these parameters It is possible to get information about each point of a graph When a mouse is moved in the Graphs area a small circle shows on the graph A coordinates hint shows above it When you hold Shift and click on a graph the circle and the hint locks i a i 101205 0 0813397 118893 0 0052356 To remove it click on the hint Also you can delete all labels by Graph gt Delete all labels context menu To select all extremum points use the Graph gt Select all extremum points context menu item All graphs are always aligned to the range shown in the Sequence zoom view It means that if you change the visible range in the overview either by zooming or scrolling the graph will also be updated The minimum and maximum values of the visible range are shown at the right lower and upper corners of the graph To close a graph uncheck its item in the popup menu e Description of Graphs e Graph Settings e Saving Graph Cuttoffs as Annotations Description of Graphs Find below the detailed description of each graph Note that characters A C G and T in the formulas denote the number of
105. R ic LEON E ESC AE Mette Gye es a eon area Ba pret Ae FOE fas tae oe SMa That os RE te Aer Yet gta 40k ee Le AS che itt ile Paes Ba ee aber IN of re fo ge oe Y rs tat di te aiee Ol wi T Pont Soy Gee ea uuen a e a area gee it o Ur esi eae EE So SER ae 3 pee TE ope eee oa a ec ee a Oh ek z ee ye Oe i rum Fat ra Li r Paa Pae el e aa at all a z 20k n cy Or EA F i 1 a eB i a a nn gt om eit at M han a eats at Cate T Tagel ta ptt liam Ly ee Fiat oe ay 2 oe Ae A T ea he Tae a el Sees ee caer se a a Vahl at or Ble a Pao rs Sr PERE vial ae T oo ior pecs dh an te a Hemati Bo a sae Ae aL Sir ape De W aE moe od A Wi a seu tee fete ie es eee ee a Se i ee ee h EL fai 60k Prue raK ee Eoaea Cope aot ee AT Ache Pri D Beal z k a n a i a a A ade a P Beef tty e teen acs E An a enga oe ATE Bb NM oft ple ate bees sot es ED TS aS fas Che See TE A ew aes oo E x 2 d an E od Be ton oe oO E Lava row on eo ee mee J ri i r Aag a a con pte 1 ve grt 0 a 1 ee 1 e TOk a Baa oo o be ak J AERO ey Ree ena ee a Pe oe a ae a ome of ts C eee SOM Te ce ee ara eee a eee eee ak rere by kai cee ae g E oe ot Ce en hele eT i Se 1 ak Cia Cae i ell a i E a rad tag alg Se A SAL ne DC ee ee A T rts as iets Le Poo cee no yo eto ue He toe Bo ei nee re oe oe th er ey eg ee a oe Dan of fees fy att lg lat es Tee gt ng es Pita m TE cote e E
106. S with position weight matrices PWM converted from input position frequency matrices PFM and saves the regions found as annotations Parameters seg semicolon separated list of input sequence files to search TFBS in String Required matrix semicolon separated list of the input PFM String Required out output Genbank file name name of the annotated regions String Optional Default misc_feature type type of the matrix Boolean Optional Default false The following values are available e true dinucleic type e false mononucleic type Dinucleic matrices are more detailed while mononucleic ones are more useful for small input data sets algo algorithm used to convert a PFM to a PWM String Optional Default Berg and von Hippel The following values are available e Berg and von Hippel e Log odds e Match e NLG score minimum percentage score to detect TFBS Number Optional Default 85 strand strands to search in Number Optional Default 0 The following values are available 283 Unipro UGENE Manual Version 1 20 0 e 0 both strands e 1 direct strand e 1 complement strand Example ugene pfm search seq in fa matrix MA0265 1 pfm MA0266 1 pfm out res gb Building PWM Task Name pwm build Builds a position weight matrix from a multiple sequence alignment file Parameters in semicolon separated list of input MSA files String Re
107. Start button The matrix will be created and saved If the Build weight or frequency matrix dialog was invoked from the Weight matrix search dialog then the matrix also will be chosen as the current profile Primer3 The Primer3 plugin is a port of the Primers tool It is intended to pick primers from a DNA sequence To use the Primer3 open a DNA sequence and select the Analyze Primer3 context menu item The dialog will appear 250 Unipro UGENE Manual Version 1 20 0 A Primer Designer Main General Settings Internal Oligo Penalty Weights RT PCR Sequence Quality Result Settings Excluded regions Targets Product size ranges 150 250 100 300 301 400 401 500 501 600 601 700 701 850 851 1000 Number to return 5 Max 7 stability Max repeat mispriming 12 00 Pair max repeat mispriming Max template mispriming 12 00 Pair max template mispriming Start codon position Pick left primer E Pick hybridization probe internal oligo Pick right primer or use left primer below or use oligo below or use right primer below 5 to 3 on opposite strand All available parameters are the same as in the original Primers However there is one additional feature available which is not originally a part of Primer3 tool It allows user design primers for RT PCR experiments by choosing which exons introns to span with the primer product This feature is described in detailed below When you select the parameters you can save and load settings
108. TEA eea hu beni 3 a ee AE oe a at Mere Bent 14 ue g in ated ssa met ie Vag a ee pete seg wera reed a Jre Ty ooh RE AY qe tt 1 ne a Tio eh Jr P a Yr k Sia Sa S J mi ara Tabe r oom Tee a cee ee apt L n o p E a e Maaa ae E o bed n a as H E T aL E ye mae ee oh Maga op Lor aniign E et or B e r es a a ee r a Pa a a 20k peo ni ii T Me aT i des os y a Tat ou a 4 a ehh ie eo ae De ame Lee aa 7 eee i ie Par TRR a 1 salt ys 1 Ca b a ril x a Cr M a Fy Pad te cally x wee E m Cees i See a Hape 1 Us hye Loy hon aS T Y u hea tee gtd h b aa Pet ee ee oe ove Baio ara Seo Pei bt pee Oe Te Sa be pa oe D a a On coh E eee tee a oa ear oe ae aes een eco eee et ae E eae ae Boe eee ene pees fa eee we a L T uh a_i wont Pee bey Pi tet LT otc ah eo er eee FF aee ri rar i e E a AT Apep th a E ia A HERI AY aay ee rege e Apatah iaa aaa brg ae H Die i a Pir ee Se ee tare A BrE pn TI ae ee i DE enl menpe r ig IAMA e ee ten T E S EE T me Ti 0k E tee TaT a eek LEPE eg A Gomes oly ioe E em iana r ae iera oepa baia i wed See Re te r3 a i e E EEE EA SST a EA A A fy Gad REPRE anh R AINE am Rae ETE i a a a E A En a Be E a a a Sve a Bae T EER a sea Spm a a Bag a 4 mE ta oe ee poa at so et oles GU Splice AL a ee T gee ous Fa F o oam t w amma T r eos ag i ce a aM aa E b 1 Soon te x I vate ALA a en Ae rel Bed tae e oe ee ne ade Ral eee ee LT
109. The Advanced options tab is not available when the cdd search is selected e Exporting BLAST Results to Alignment e Fetching Sequences from Remote Database 178 Unipro UGENE Manual Version 1 20 0 Exporting BLAST Results to Alignment To export BLASt results as alignment select the results in the Annotations Editor and call the Export gt Export BLAST result to alignment cont ext menu item The following dialog will appear amp Export BLAST Result to Multiple Alignment Export to file File format to use Qualifier to use as nane Add reference to alignment Add document to the project The following parameters are available Export to file name of the new file File format to use format of the new file The following formats are available CLUSTALW FASTA MSF MEGA NEXUS PHYLIP Interleaved PHYLIP Sequential Stockholm Qualifier to use as name name of the qualifier The following qualifiers are available accession def id Add reference to alignment adds a reference to alignment Add document to the project adds the new document to the project Select the options and click on the Export button Fetching Sequences from Remote Database Each result annotation found with the remote BLAST in UGENE has accession and id qualifiers that can be used to fetch the corresponding sequences from the NCBI The prompt way to fetch the sequences of several annotations is the following e Select the annotations in the
110. Transcription Factors Binding Sites e Types of SITECON Models e Eukaryotic e Prokaryotic e Building SITECON Model e Smith Waterman Search e HMM2 e Building HMM Model HMM Build e Calibrating HMM Model HMM Calibrate e Searching Sequence Using HMM Profile HMM Search e Building HMM Model HMM3 Build e Searching Sequence Using HMM Profile HMM3 Search e Searching Sequence Against Sequence Database Phmmer Search e uMUSCLE e MUSCLE Aligning e Aligning Profile to Profile with MUSCLE e Aligning Sequences to Profile with MUSCLE ClustalW MAFFT T Coffee Bowtie e Bowtie Aligning Short Reads e Building Index for Bowtie e Bowtie 2 e Bowtie 2 Aligning Short Reads Unipro UGENE Manual Version 1 20 0 e Building Index for Bowtie 2 e BWA e Aligning Short Reads with BWA e Building Index for BWA e BWA SW e Aligning Short Reads with BWA SW e Building Index for BWA SW e BWA MEM e Aligning Short Reads with BWA MEM e Building Index for BWA MEM e UGENE Genome Aligner e Aligning Short Reads with UGENE Genome Aligner e Building Index for UGENE Genome Aligner e Converting UGENE Assembly Database to SAM Format e CAP3 e SPAdes e Weight Matrix e Searching JASPAR Database e Building New Matrix e Primers e RTPCR Primer Design Spliced Alignment mRNA to genomic External Tools e Configuring External Tool Query Designer Plasmid Auto Annotation ClustalO Kalign Aligning Expert Discovery e Loading Sequences Mapping Sequences Markup Sequences
111. U during the alignment the corresponding hardware should be available on your computer e Align reverse complement reads use both a read and its reverse complement during the alignment e Use best mode during the alignment report only about best alignments in terms of mismatches e Omit reads with qualities lower than omit all reads with qualities lower than the specified value Reads that have no qualities are not omited Advanced parameters Maximum memory for short reads maximum memory usage for short reads This parameter allows one to decrease the load on the computer on one side and to increase the computer speed of the task on the other side e Total memory usage shows the total memory usage e System memory size shows the total system memory size Index parameters Reference fragmentation this parameter influences the number of parts the reference will be divided It is better to make it bigger but it influences the amount of memory used during the alignment e Index memory usage size shows the index memory usage e Directory for index files temporary directory for saving index files You can choose a temporary directory for saving index files for the reference that will be built during the alignment If you need to run this algorithm one more time with the same reference and with the same reference fragmentation parameter you can use this prebuilt index that will be located in th
112. UGENE NC_014267 1 s NC_014267 Eal File Actions Settings Tools o A Gf B amp B Go a E Aa v 11 Ss ix ah ost Restriction Sites Map BamHI 0 sites Bglll 0 sites Clal 0 sites Dral 0 sites EcoRI 0 sites Hindi 0 sites PstI 0 sites Sall 0 sites Smal 0 sites Xmal 0 sites trnc YP _003734611 1 1 Project 4 i human_Tl fa a s human_T1 UCSC 4 i NC_014267 1 gb a NC_014267 featu Q s NC_014267 W human _T1 s human_T1 vy NC_014267 1 s NC_014 g Auto annotations NC_014267 1 gb NC_0142 gt g NC_014267 features NC_014267 1 gb nm To load a saved project later select File Open and specify the path to the project file Search in Project Use the search field in the project view to search in the whole project FASS4 6 R12 1 413 324 a FASS 6 R121 49 94 a 6_R1_2_1_540_792 A meu UCSC April 2002 chr7 115977709 117855134 human _T1 fa NC_001363 features 4 CDS cy san a features d comment ey Ne Ot ras features 4 source 163 features aig Options Panel The Options Panel is available in the Sequence View and in the Assembly Browser By default it is closed To open a tab of the Options Panel click on the corresponding icon at the right side of a Sequence View or Assembly Browser window To close the tab click again on the 34 Unipro UGENE Manual Version 1 20 0 tab icon More
113. Unipro AGENE Unipro UGENE Manual Version 1 20 0 December 16 2015 Unipro UGENE Manual Version 1 20 0 Unipro UGENE Online User Manual About Unipro About UGENE Key Features User Interface High Performance Computing Cooperation Download and Installation System Requirements UGENE Packages Installation on Windows Installation on Mac OS X Installation on Linux e Native Installation on Ubuntu e Native Installation on Fedora e Basic Functions UGENE Terminology UGENE Window Components Welcome Page Project View Task View Log View e Notifications Main Menu Overview Creating New Project Creating Document Opening Document e Opening for the First Time e Advanced Dialog Options e Opening Document Present in Project e Opening Several Documents Opening Containing Folder Exporting Documents Locked Documents Using Objects and Object Views Exporting Objects e Exporting Sequences to Sequence Format Exporting Sequences as Alignment Exporting Alignment to Sequence Format Exporting Nucleic Alignment to Amino Translation Export Sequences Associated with Annotation Using Bookmarks Exporting Project Search in Project Options Panel Adding and Removing Plugins Searching NCBI Genbank Fetching Data from Remote Database UGENE Application Settings General Resources Network File Format Directories Logging Alignment Color Scheme External Tools Settings Genome Aligner Workflow Designer Settings OpenCL e S
114. Unipro UGENE Manual Version 1 20 0 m Align with Clustal Omega Iteration L Number of iterations Max number guidetree iterations Max number of HMM iterations Miscellaneous Number of CPUs being used Set options automatically Kalign Aligning Kalign is a fast and accurate multiple sequence package designed to align large numbers of protein sequences Kalign home page KAlign To use Kalign open a multiple sequence alignment file and select the Align with Kalign item in the context menu or in the Actions main menu The following dialog appears Align with Kalign Advanced options Gap open penalty Gap extension penalty Terminal gap penalty F Bonus score Translate to amino when aligning Output file 1 The Standard Genetic Code The following parameters are available Gap opening penalty indicates the penalty applied for opening a gap The penalty must be negative Gap extension penalty indicates the penalty applied for extending a gap Terminal gap penalty the penalty to extend gaps from the N C terminal of protein or 5 3 terminal of nucleotide sequences Bonus score a bonus score that is added to each pair of aligned residues Translate to amino when aligning translates an alignment to amino when aligning Expert Discovery ExpertDiscovery system applies an original knowledge discovery approach Relational Data Mining Scientific Discovery Web Site Vityaev 2006 Vityaev Kovalerchuk
115. UrcE c L E C p m K R m g Show all annotation names 4 l j Configure the annotations Show annotations a cy MC 001363 features m gt a source 0 1 l ve misc_feature 0 2 a E misc_feature Misc Feature Show on translation Show value of qualifier m 5245 5833 Se note Show value of qualifier gt D misc feature Misc Feature 2 990 ve comment 0 1 a cs oy gt E CDS CDS 5048 5203 a E CDS CDS 3875 4999 ii If you input several qualifiers names separated by comma then the first found qualifier is taken into account and shown on the annotation Creating and Editing Qualifier To add a qualifier to an annotation select it in one of the Sequence View subviews and press the Insert key or use the Add Qualifier context menu or the Actions main menu item e S UTR Select 405uT m eae Add 4 New annotation Ctril N a W cos Analyze Objects with annotations 4B cps Align r Ins cod Cloning db 3 Export p 50124074 db i inelD 1489680 Edit j ge Lab locu Remove p noti was assumed that the SARS orflab polyprotein Rulers rol e enna flab polyprotein pplab protein_id MP 828849 2 The dialog will appear 73 Unipro UGENE Manual Version 1 20 0 G4 Add New Qualifier Name new _qualifier Here you can specify the name and the value of the qualifier You can use the F2 key to rename a qualifier Ea Rename Qualifier 2
116. Use this tab to configure the Workflow Designer settings 44 Unipro UGENE Manual Version 1 20 0 Application Settings General oe Resources Network Dan File Format Show grid Logging Alignment Color Scheme 1 Snap to grid External Tools Element style Genome Aligner OpenCL Element font Runtime settings Track running progress Enable debugger Directories Use directory for output files Directory for custom elements with scripts Directory for custom elements with command line tools Directory for induded schema elements SSS ow m i OpenCL If you have a video card that supports OpenCL you can use it to speed up some calculations in UGENE To do it install the latest video card driver and check the corresponding check box Application Settings General OpenCL Resources Network The following OpenCL enabled GPUs are detected File F P Check the GPUs to use for accelerating algorithms computations rile Formal Logging Intel R Corporation Intel R HD Graphics 4600 1297 Mb Alignment Color Scheme NVIDIA Corporation GeForce GT 740M 2048 Mb External Tools Genome Aligner OpenCL Workflow Designer Now you can for example use OpenCL optimization for the Smith Waterman algorithm 45 Unipro UGENE Manual Version 1 20 0 Sequence View Sequence View Components Global Actions Sequence Toolbars Sequence Overview Sequence Zoom V
117. Version 1 20 0 General Logging Resources Network Category TRACE DETAILS INFO ERROR lt lt all gt gt E Sample text Sample text Sample text Sample text File Format Logging Alignment Color Scheme External Tools Console Genome Aligner OpenCL Workflow Designer Algorithms Core Services Input Output Performance Remote Service Scripts Tasks Teamcity Integration Teamcity Log User Actions User Interface Eee hehehehenenene a Gee e oa aaa a See ee eee SICH CH CH CHC a a a Go a a Ss Sa Log format W Show date Show log level Show log category Enable colored log output Save output to fles Date format hh mm On the Logging tab you can select type of log information ERROR INFO DETAILS TRACE for each Category that will be output to the Lo g View You can select format for each log message by checking the Show date Show log level and Show log category options Log x NF0 09 59 Starting Open new Sequence view task r INF0 09 59 Task Open new Sequence view finished TNFO 09 59 Starting Open new Sequence view task INFO 09 59 Task Open new Sequence view finished J INFO 09 59 Starting Open new Sequence view task NFO 09 59 Task Open new Sequence view finished Alignment Color Scheme mnsa a General Resources Network File Format Logging Alignment Color Scheme External Tools Genome Aligner OpenCL Workflow Designer A
118. View and click Enter double click on it or drag it to an empty space of the UGENE window Opening Several Documents To open several documents that are not yet presented in the current project use the File Open item in the main menu The Select files open dialog will appear Select the documents with a help of the Ctrl button and click on the Open button The following dialog will appear m sequence Reading Options The document selected contains multiple sequence instances Please select the way UGENE will read these sequences As separate sequences in sequence viewer 7 Merge sequences into a single sequence to show in sequence viewer Number of unknown symbols W for quoleic ar X for amino between parts 10 bases 9 Join sequences into alignment and open in multiple alignment viewer Align reads to reference sequence File preview gt d GACTAGC gt a GACTAGC gt E GACTAGC 24 Unipro UGENE Manual Version 1 20 0 Select the reading options and click on the OK button Opening Containing Folder To open a containing folder of the document that is already present in the current project select it in the Project View and click on the Open containing folder context menu item Exporting Documents lf a document has a format that supports writing in UGENE see the Supported File Formats chapter you can export the document to a new document in a required format To do it use the Export document it
119. a Code 5 The Invertebrate Mitochondrial Code 6 The Ciliate Dasycladacean and Hexamita Nuclear Code 9 The Echinoderm and Flatworm Mitochondrial Code 10 The Euplotid Nuclear Code 11 The Bacterial and Plant Plastid Code 12 The Alternative Yeast Nuclear Code 13 The Ascidian Mitochondrial Code 14 The Alternative Flatworm Mitochondrial Code 15 Blepharisma Nuclear Code 16 Chlorophycean Mitochondrial Code 21 Trematode Mitochondrial Code 42 Scenedesmus obliquus Mitochondrial Code 23 Thraustochytrium Mitochondrial Code The codon table will appear 174 Unipro UGENE Manual Version 1 20 0 W UGENE human_T1 s human_T1 UCSC Apnil 2002 chr 115977709 117855134 p File Actions Settings Tools Window Help moB A a a h ge oe a C A G ener UUU unten UCU UAU UGU u E m uUUC a UAC UGC c UUA UAA UGA EEE A neg UUG uce UAG UGG Tryptophan Trp W G l am CAU Histidine His H SY 2 c aa CGC arginine Arg R A CUG CAG G g r AUC AAC c PUA DAR Arginine Arg R i AUG a Aes AAG G GUU GAU U a GUC or GAC C GUA GCA GAA A GUG GCG GAG G Clicking on a codon name redirects you to Wikipedia to give you a brief description of the corresponding amino acid Cells of the table are colored according to classes of amino acids Remote BLAST The Remote BLAST plugin provides a capability to annotate sequences with information stored in the NCBI BLAST remote database To perform a remote database se
120. a fragment from a DNA molecule from the current UGENE project Click the From Project button to do so The Select Item dialog appears with the sequence objects available Select a sequence and press the OK button After that create a fragment in the appeared Create DNA Fragment dialog as described in the Creating Fragment paragraph The fragment created from the sequence appears in the list of available fragments Fragments of the New Molecule The next step is to add required fragments to the new molecule contents To add fragments select them in the list of available fragments and click the Add button or by double click on a fragment To add all the fragments click the Add All button Changing Fragments Order in the New Molecule To change the order of fragments in the new molecule select a fragment in the new molecule contents list and click either the Up or the Down button to move the fragment in the corresponding direction Removing Fragment from the New Molecule 195 Unipro UGENE Manual Version 1 20 0 To remove a fragment from the new molecule select it in the new molecule contents list and click the Remove button To remove all the fragments click the Clear All button Editing Fragment Overhangs To edit a fragment s overhangs select the fragment in the new molecule contents list and click the Edit button The Edit Molecule Fragment dialog appears E Edit Molecule Fragment Left End Type Overhang Blunt Custom o
121. abase Database in the Project Deleting Data Drag n drop in the Database Exporting Objects from the Database Configuring Database 263 Unipro UGENE Manual Version 1 20 0 To make use of a shared database follow the steps below 1 Deploy a MySQL database server We recommend you to download MySQL binaries from the official site Note that UGENE supports MySQL versions 5 5 and higher Here you can also find instructions on how to install and launch a MySQL server instance for each platform 2 Create an empty database Log in to the MySQL server as a user with administrative privileges you must be able to create databases and users and to grant privileges to the created users In the MySQL console or in your favorite SQL browser execute the following command gt CREATE DATABASE your_database_name 3 Create database users You may probably want to limit possible influence on the shared database by the UGENE users who will use it In this case create a distinct MySQL user for each UGENE user or a group of users In order to do this execute the following commands gt CREATE USER user_nickname IDENTIFIED BY user_password Decide whether the created user is allowed to modify the database content or only to view it In the first case execute the command below gt GRANT CREATE SELECT INSERT INDEX UPDATE DELETE CREATE ROUTINE EXECUTE DROP ON your_database_name TO user_nickname IDENTIFIED BY
122. acia aes Selected enzymes ed enzymes Load selection BamHI BbsI Bgl Clal Dral EcoRI HindIII PstI Sall Smal Xmal REBASE Info Filter by number of results Minimum hits Maximum hits Exclude region Region Whole sequence Total number of enzymes 4862 selected 11 You can see the list of restriction enzymes that can be used to search for restriction sites The information about enzymes was obtained from the REBASE database For each enzyme in the list a brief description is available the accession ID in the database the recognition sequence etc If you re online you can get more detailed information about an enzyme selected by clicking the REBASE Info button Selecting Restriction Enzymes Using Custom File with Enzymes Filtering by Number of Hits Excluding Region Circular Molecule Results Selecting Restriction Enzymes To select an enzyme check it in the list Notice that the enzyme appears in the Selected enzymes area of the dialog You can also use the Select Al button to select all the enzymes available the Select None button to deselect all the enzymes To select all enzymes with recognition sequence length shorter than the specified value click the Select by length button and input the minimum length in the dialog appeared 191 Unipro UGENE Manual Version 1 20 0 To invert selection click the nvert selection button As soon as enzymes are selected you can click the OK button to search for corre
123. ads is 251 a ak ok q 1k 9 168 C251 9 3k ib e inne _ To show hide the coordinates on the ruler you can click the following button on the toolbar Tt k To show hide the coverage on the ruler you can click the following button on the toolbar Cio0 Alternatively you can use the Show coordinates and Show coverage under cursor check boxes located on the Assembly Browser Settings ta b of the Options Panel Go to Position in Assembly To go to the required position in an assembly use the following field located on the Assembly Browser toolbar Input the location and click the Go button A similar Go field is also available on the Navigation tab of the Options Panel Using Bookmarks for Navigation in Assembly Data Use bookmarks to save and restore visual state of an assembly for example position in the assembly zoom scale etc Getting Information About Read A read displayed in the Reads Area consists of the bases A C G T It may also contain the N character that stays for an ambigous base Depending on the value of the Cigar parameter the read can be shown partially or gaps can be inserted inside the read see below By default when a read is hovered over in the Reads Area a hint appears 146 Unipro UGENE Manual Version 1 20 0 To disable this behaviour click the following button on the toolbar Or uncheck the Show pop up hint check box on the Assembly Browser Settings tab of the Options Panel The hint s
124. age formats You can export whole alignment or custom region To select the custom region click on the Se ect button Statistics To show statistics use the Statistic tab of the Options Panel Y Reference sequence Select and add x Y Distances column Show distances column Automatic updating Press button to update Fy Hint select a reference above Here you need to select a reference sequence Also you can change the distance algorithm select the profile mode and exclude gaps To generate distance matrix and grid profile see the documentation below 133 Unipro UGENE Manual Version 1 20 0 e Distance Matrix e Grid Profile Distance Matrix Using the Alignment Editor you can also create a distance matrix of a multiple sequence alignment To create a distance matrix use the Statistics Generate distance matrix item in the Actions main menu or in the context menu The dialog will appear Ss Generate Distance Matrix Distance algorithm Hamming dissimilarity Profile mode Counts 9 Percents Exdude gaps Show group statistics of multiple alignment Save profile to file File Hypertext HTML Comma separated CSV The following parameters are available Distance algorithm there are two distance algorithms Hamming distance for dissimilarity and Simple similarity for similarity Profile mode Counts Percents select the Percents to have scores shown as percents in the report Also you
125. airs Boolean VW A C G T only Number Example ugene snp bam test bam ref test_ref fa wout test_out vcf Generating DNA Sequence Task Name generate dna Generates a random DNA sequence with specified nucleotide content Parameters algo Algorithm for generating using GC Content by default String content Specifies if the nucleotide content of generated sequence s will be taken from reference or specified manually A G C T parameters using manual by default String count Number of sequences to generate using 1 by default Number length Length of the resulted sequence s using 1000 bp by default Number a Adenine content using 25 percents by default Number c Cytosine content using 25 percents by default Number g Guanine content using 25 percents by default Number t Thymine content using 25 percents by default Number ref Path to the reference file could be a sequence or an alignment String seed Value to initialize the random generator By default seed 1 the generator is initialized with the system time using 1 by default Number wna size Size of window where set content using 1000 by default Number accumulate Accumulate all incoming data in one file or create separate files for each input In the latter case an incremental numerical suffix is added to the file name using True by default Boolean format Output file form
126. al Profile for SITECON Task Name sitecon build Builds a statistical profile for SITECON It can be later used to search for TFBS Parameters in semicolon separated list of input DNA multiple sequence alignment files An input file must not contain gaps String Required out output file If several input files have been supplied then a sitecon profile is built for each input file i e several output files with different indexes are generated String Required wsize window size The window is a region of the alignment used to build the profile It is picked up from the center of the alignment and occupies the specified length The edges of the alignment beyond the window are not taken into account The recommended length is a bit less than the alignment length but not more than 50 bp Number Optional Default 40 clength length of a random synthetic sequence used to calibrate the profile Number Optional Default 1000000 rseed random seed used to calibrate the profile e g to generate the random synthetic sequence Use the same value to get the same calibration results twice on the same data By default new random seed is generated each time a calibration occurs Number Optional Default 0 walg specifies to use the Algorithm 2 weight algorithm In most cases it is not required but in some cases it can increase the recognition quality Boolean Optional Default false Example ugene sitecon buil
127. align short reads to This parameter is required Result file name file in SAM format to write the result of the alignment into This parameter is required Library single end or paired end reads 225 Unipro UGENE Manual Version 1 20 0 Prebuilt index check this box to use an index file instead of a source reference sequence The index is a set of 6 files with suffixes 1 ebwt 2 ebwt 3 ebwt 4 ebwt rev 1 ebwt and rev 2 ebwt The index is created during the alignment Also you can build it manually SAM output always save the output file in the SAM format the option is disabled for Bowtie Short reads each added short read is a small DNA sequence file At least one read should be added A Short reads length for Bowtie can t be more than 1024 You can also configure other parameters They are the same as in the original Bowtie you can read detailed description of the parameters on the Bowtie manual page Select one of the following alignment modes The n alignment mode When the n mode is selected Bowtie determines which alignments are valid according to the following policy Alignments may have no more than N mismatches where N is a number 0 3 in the first L bases where L is a number 5 or greater set with Seed length on the high quality left end of the read The sum of the Phred quality values at all mismatched positions not just in the seed may not exceed E set with Maq error Where qualities are u
128. ameters are set click the Export button The consensus sequence is exported to the file and if the Add to project check box has been checked it is added to the current project and opened Exporting Consensus Variations To export a consensus sequence variations of the assembly select the Export consensus variations item in the Consensus Area context menu The following dialog will appear 152 Unipro UGENE Manual Version 1 20 0 m Export Consensus Vanations Export to file reformat Consensus algoritm Keep gaps Add to project Select a file mode and the file format The following modes are available Variations Similar and All Variations can be exported as to a SimpleSNP or VCFv4 file Modify if required the consensus algorithm The consensus is exported with gaps if the Keep gaps check box has been checked Also you can select the exporting region It can be either a Whole sequence a Visible region or a Custom region When all the parameters are set click the Export button The consensus sequence is exported to the file and if the Add to project check box has been checked it is added to the current project and opened i The Export consensus variations feature is available when the reference sequence is associated with assembly Exporting Assembly as Image To export the visible part of the assembly as an image select either the Actions Export as image item in the main menu or the following button on the toolba
129. ample reporting thresholds options can be configured using the dialog 216 Unipro UGENE Manual Version 1 20 0 M HMM3 Search Reporting thresholds Report domains with E value less than Report domains with score greater than Score threshold Use profile s GA gathering cutoffs Use profile s NC noise cutoffs Use profile s TC trusted cutoffs E Number of significant sequences for domain E value calculation 0 00 The search results are stored as sequence annotations in the Genbank file format cy gi 2136280 pir 138344 titin human l 200k 400k 600k 800k im 12m 14m 16m 16m am 223m 24m 26m 26m 4 aT 1 TOIS s PAK cece IDS eee Ah S CS ee PO es a i A T140 PIAS Ab eee 4 El Annotations MyDocument_3 gb G2 hmm_signal 0 24024 EH E hrani_signal 6594 6679 EH E hrm_signal 6695 6781 EH E hrm_signal 6796 6882 EH E hrm_signal 6992 7076 E hrm_signal 7092 7177 oa Accuracy per residue 9 7 6351e 01 Blas 3 53754e 02 Conditional e value 5 96204e 17 been Envelope of domain location O91 7177 HMM model faa Accession number in PFAM database PFOOO41 H HMM region 1 87 been Independent value 1 69874e 17 es Score 49 864132 EH E hmm signal 7208 7372 EH E hmm signal 7387 7473 Q The HMM3 search works only with files that contain a single HMM model Searching Sequence Against Sequence Database Phmmer Search The Phmmer search tool searches for query sequence matches in sequence
130. annotations String Required name name of the annotated regions String Optional Default misc_feature ptrn subsequence pattern to search for e g AGGCCT String Required score percent identity between the pattern and a subsequence Number Optional Default 90 matrix scoring matrix String Optional Default Auto Among others the following values are available e blosum62 e dna e rna e dayhoff e gonnet e pam250 e etc The matrices available are stored in the UGENE data weight_matrix directory filter results filtering strategy String Optional Default filter intersections The following values are available e filter intersections none Example ugene find sw in human_Tl fa out sw gb ptrn TGCT filter none Adding Phred Quality Scores to Sequence Task Name join quaility Adds Phread quality scores to a sequence and saves the result to the output FASTQ file Parameters in input sequence file String Required quality input Phred quality scores file String Required out output FASTQ file String Required Example ugene join quality in e_coli fa quality e_coli qual out res fastg Local BLAST Search Task Name local blast Performs a search on a local BLAST database using old version of the NCBI BLAST Q BLAST is used as an external tool and must be installed on your system Parameters toolpath path to the blas
131. antha_deracan ychia_baranovi ettigonia_viridissima onocephalus_discolc onocephalus_sp onocephalus_percat lecopoda_elongata_ lecopoda_elongata_ lecopoda_sp __ Male odisma_sapporensis etrodes_pupus EFS haneroptera_falcatz ae ee S S S S S S S S S Scale range 0 011 Font size a Line width 1 Branches width Height _ To load a tree from a file follow the instruction described in the Opening Document paragraph or use the Tree settings tab of the Options Panel For example you may open the UGENE data samples Newick COI nwk sample file provided within UGENE package To build a tree from a multiple sequence alignment see the Building Phylogenetic Tree paragraph To learn what you can do with a tree using UGENE Phylogenetic Tree Viewer read the documentation below e Tree Settings e Selecting Tree Layout and View e Modifying Labels Appearance e Showing Hiding Labels e Aligning Labels e Changing Labels Formatting e Adjusting Branch Settings Zooming Tree Working with Clade e Selecting Clade Collapsing Expanding Branches Swapping Siblings Zooming Clade Adjusting Clade Settings Changing Root e Exporting Tree Image e Printing Tree Tree Settings To adjust a tree settings select either the Tree Settings toolbar button or the Tree settings tab of the Options Panel The Tree settings tab 157 Unipro UGENE Manual Version 1 20 0 Show nam
132. ap size AtS side bp 5 lt At 3 side bp 5 Exon range v Primer product must span at least one intron on the corresponding genomic DNA a Max number of pairs to query 1000 Region Whole sequence z 1 199950 Save settings Load settings Reset form Pick primers The following parameters are available Exon annotation name To detect exon boundaries UGENE searches for exonic annotations This option allows to set custom name for annotations denoting exons Default value is exon Minimum exon junction overlap size If checked then only the pairs with at least one of the primers overlapping exon junction in the mRNA sequence will be selected At 5 side bp Minimum overlap size on the 5 side of the exon junction Default is 5 bp At 3 side bp Minimum overlap size on the 3 side of the exon junction Default is 5 bp Exon range This option allows to limit the sequence region where the primers are searched for For example setting value 3 5 will limit the search to a sequence region consisting of exons 3 4 5 of the transcript as defined by the order in the sequence Default value is an empty string which means that there are no limitations Span at least one intron This option makes sure that primer product should span an intron on the genomic sequence i e the forward and reverse primers must be located in different exons The option is enabled by default Max numbers of pairs
133. aq rounding nomaground Maq Mapping and Assembly with Quality accepts quality values in the Phred quality scale but internally rounds values to the nearest 10 with a maximum of 30 By default Bowtie also rounds this way No Maq rounding prevents this rounding in Bowtie No forward orientation nofw do not attempt to align against the forward reference strand No reverse complement orientation norc do not attempt to align against the reverse complement reference strand Try as hard tryhard try as hard as possible to find valid alignments when they exist including paired end alignments Best alignments best make Bowtie guarantee that reported singleton alignments are best in terms of stratum i e number of mismatches or mismatches in the seed for the case of n mode and in terms of the quality values at the mismatched position s All alignments all report all valid alignments per read or pair Validity of alignments is determined by the alignment policy combined effects of n mode v mode Seed length and Maq error Select the required parameters and press the Start button Building Index for Bowtie To build Bowtie index select the Tools DNA Assembly Build index item in the main menu The Build Index dialog appears Set the Align short reads method parameter to Bowtie 226 Unipro UGENE Manual Version 1 20 0 The dialog looks as follows H Build Index Align short reads met
134. arch open a Sequence View select a sequence region to analyze and click the Analyze Query NCBI BLAST database context menu item If a region is not selected the whole sequence will be analyzed Goto position 3 ed Select sequence region tr ACG Find pattern Ctrl F HM A New annotation r W Find pattern Smith Waterman Ctrl Shift F EE Rename item Mo Find ORFs 4 x ae Find annotated Lai my Select o 9 Build dotplot J Add Find repeats Analyze Find tandems Align Analyze with query schema Cloning se Find restriction sites Export E Query NCBI BLAST database Edit sequence t Ha Search HMM signals with HMMER3 The following dialog will appear where you can choose the search options 175 Unipro UGENE Manual Version 1 20 0 Ca search Through a Remote Database General options Advanced options Select the search type blastn C Search for short nearly exact matches Expectation value 10 000000 E Megablast Results limit 20 The database The database description Basic Local Alignment Search Tool or BLAST is an algorithm for comparing primary biological sequence information such as the amino acid sequences of different proteins or the nucleotides of DNA sequences A BLAST search enables a researcher to compare a query sequence with a library or database of sequences and identify library sequences that resemble the query sequence above a certain threshold w Save anno
135. at using fasta by default String split Split each incoming sequence on several parts using 1 by default Number out Output file String Example ugene generate dna length 2000 a 45 out test fa Creating Custom CLI Tasks The predefined tasks are actually the Workflow Designer schemas stored in the UGENE data cmdline directory Follow the instructions in the Workflow Designer Manual on how to create a schema and to run it from the command line You may also find useful the following video tutorial devoted to the creating of a custom console command e Creating custom console command MUSCLE alignment with various output format 289 Unipro UGENE Manual Version 1 20 0 APPENDIXES e Appendix A Supported File Formats e Specific File Formats e UGENE Native File Formats e Other File Formats Appendix A Supported File Formats i UGENE is able to read and write files compressed with Unix Linux gzip utility You don t have to unpack the files e Specific File Formats e UGENE Native File Formats e Other File Formats Specific File Formats File format File extension Read Write Comment ABIF ab1 abi abif A chromatogram file format See also Chromatogra m Viewer ACE ace A file format for storing data about genomic contigs See also Alignment Editor Bairoch bairoch A file format to store enzymes See also Restriction Analysis BAM bam Binary compressed SAM
136. atically when you open a PDB or MMDB file For example open UGENE data samples PDB 1CF7 PDB The 3D Structure Viewer adds a view to the upper part of the Sequence View ft 3D Structure Viewer Active view l 1CF7 Display Links ACF chain 1 sequence amino ae 3 1 fF i2 4 6 amp 10 12 14 16 18 20 22 24 26 26 30 32 M J 3 40 42 44 46 46 50 52 M 56 58 60 62 64 57 SRHEKFSLGELLTITRFVSLLQ EAKDGCVLDLELAADTLAVEAQGERRIYDITNVLEGIGLIERKSENSIQWE y a I r Notice the Links button on the toolbar When you click the button the menu appears with quick links to online resources with detailed information about the molecule opened PDB Wiki RSCB PDB PDBsum NCBI MMDB Note that if you re online you can access the Protein Data Bank directly from UGENE and load a required file by its PDB ID see Fetching Data from Remote Database for details Hint Don t forget to select the correct database PDB while fetching Changing 3D Structure Appearance This chapter describes how you can change a 3D stucture appearance Selecting Render Style Selecting Coloring Scheme Calculating Molecular Surface Selecting Background Color Selecting Detail Level Enabling Anaglyph View Selecting Render Style The following render styles are available e Ball and Stick e Space Fill 90 Unipro UGENE Manual Version 1 20 0 e Tubes e Worms To change the render style select an appropriate item in the Render Sty
137. atively maybe you would like to analyze a certain sequence part In this case you select the required data in the web browser window the Open selected in UGENE item should now appear in the context menu 298 eh Ensembl genome browser W Unipro UGENE Manual Version 1 20 0 gt OE wewensembl org Homo_sapiens Export Output Gene db core flank3_display 0 flank5_disp JJ wl Login Register Sess a ee eae E Search Human q Human GRCh37 w Location 2 145 524 002 145 337 001 Gene ACO10090 1 Transcript ACO10090 1 001 Gene based displays Gene summary Splice variants 1 Supporting evidence sequence External references Regulation Comparative Genomics Hl f Genomic alignments E Gene tree image Gene tree text Gene tree alignment Gene gain loss tree Orthologues Paralogues Protein families Phenotype E Genetic Variation Variation table Variation image structural variation E External data L Personal annotation E ID History L Gene history 4 Configure this page gy Add your data ch Export data Ft Bookmark this page lt Share this page The selected data will be opened in UGENE Export Gene Data PENSGOO000232606 ENSTOCO00413525 cdna KNOWN lincRNA AGCTTCACATGTGAGATAAATGCACTCAAAGATTCCTCACAAGTAGCTCTITGGAGCTIC AGATGTGAAATGGATCATICCTCAATCTGTIAATAGACCCTICTGIGAAGCTCITCAATCA AACCAGAGAATTCAAGAGITICCAACACCTAAGAGIGGTATITGGOCASATGGIGGGCCAA AGGAATASAGAAGGCATGCAASACTCTIGACAGAAGACATTCAGAAATTIGAT
138. average_threshold average window threshold in the area i e total_ threshold windows_number e total_threshold sum of all window thresholds in the area e windows_number number of windows in the area Ml dna_flex 144 156 E area_average_threshold total_threshold windows number i Using the DNA Graphs Package you can see the flexibility graph of a DNA sequence DNA Statistics The DNA Statistics plugin provides exportable statistic reports In the current UGENE version the DNA Statistics plugin provides only Alignment Grid Profile report The Alignment Grid Profile shows positional amino acid or nucleotide counts highlighted according to the frequency of symbols in a row The original idea of the MSA Grid Profile is described in the following paper Alberto Roca Albert Almada and Aaron C Abajian ProfileGrids as a new visual representation of large multiple sequence alignments a case study of the RecA protein family BMC Bioinformatics 2008 9 554 Usage example 169 Unipro UGENE Manual Version 1 20 0 Open a sequence alignment in the Alignment Editor and use the Statistics Generate grid profile context menu item i F Go to E E Chilo d EE 14i Edit 14i fag Alon MH SHA ey int f View 7 iat _ Advanced i TW The dialog will appear 2 Generate Alignment Profile Profile mode Counts Percents Custom options Show scores for gaps E
139. aviour Emulate hmms behaviour Emulate hmmew behaviour i The HMM build tool does not automatically calibrate a profile Use the HMM calibrate tool to calibrate the profile Calibrating HMM Model HMM Calibrate The HMM calibrate tool reads a HMM profile file scores a large number of synthesized random sequences with it fits an extreme value distribution EVD to the histogram of those scores and re saves the hmm file including the EVD parameters To avoid modification of the original HMM file you can select a new location for the calibrated profile U HMM Calibrate HMM file E Expert options Fix the length of the random sequences to Mean length of the synthetic sequences Number of synthetic sequences sooo Standard deviation 200 00 Random seed E Save calibrated profile to file Searching Sequence Using HMM Profile HMM Search The HMM search tool reads a HMM profile from a file and searches the sequence for significantly similar sequence matches The sequence must be selected in the Project View or there must be an active Sequence View window opened If the selected sequence is nucleic and the HMM profile is built for amino alignment the sequence is automatically translated and all 6 translations are used to search in If a HMM profile is built for nucleic alignment the search is performed for both strands direct and complement 213 Unipro UGENE Manual Version 1 20 0 Wy HMM Search File wit
140. bar e File Open item in the main menu or drag the file to the UGENE window Also it is able to drag and drop documents not objects between opened UGENEs Documents created not by UGENE are locked To be able to edit the document you should save a copy of the document and continue working with the copy e Advanced Dialog Options Advanced Dialog Options Open the Select Correct Document Format dialog by Add Existing document item in the Project View context menu or by File Open As item in the main menu The foolowing dialog will appear 23 Unipro UGENE Manual Version 1 20 0 T Select Correct Document Format Options for human_T1 fa FASTA format Score 13 Perfect match Plain text format Score 2 ow similarity BED format Score 2 low similarity O Raw sequence format Score 1 tery bow similarity O MSF format Score 1 bery dow similarity Choose format manually File preview shuman_T1 UCSC April 2002 chr 115977709 117855134 TIGTCAGATTCACCAAAGTIGAAATGAAGGAAAAAATGCTAAGGGCAGCC AGAGAGAGGTCAGGTTACCCACAAAGGGAAGCCCATCAGACTAACAGCGG ATCTCTCGGCAGAAACCCTACAGGCCAGAAGAGAGTGGGGGCCAATATTIC CATATTCTTAAAGAAAAGAATITTICAACCCAGAATITCATATCCAGCCAA Here you can choose how to interpret the data stored in the file The format is detected automatically but you can select it manually Opening Document Present in Project To open a document that is already present in the current project select it in the Project
141. ce View and select the Analyze Find repeats context menu item a mmr Analyze F R Find pattern Ctrl F Align P SH Find pattern Smith Waterman Ctrl Shift F Cloning F Find ORFs Export t Find annotated regions Edit sequence a Annotate plasmid and custom features Remove J Build dotplot Find repeats Al Rulers Find tandems alk eeN gf Analyze with query schema The dialog will appear that allows specifying repeat parameters and the annotations table document to save the results into 186 Unipro UGENE Manual Version 1 20 0 I Find Repeats Repeat finder parameters Window size 100bp Minimum indentity per window 100 Minimum distance between repeats Maximum distance between repeats Region to process 7 Save annotation s to Existing table F NC_014267 features NC_014267 1 ab Create new table Use auto annotations table Y Annotation parameters Group name lt auto gt Annotation name repeat_unit Description Estimated repeats count 0 The dialogues status line displays approximate repeats number that will be found with the current settings The Advanced tab provides additional repeats finding options 187 Unipro UGENE Manual Version 1 20 0 0 Find Repeats Advanced parameters Custom algorithm E Search only for repeats that lie inside of an annotated region E Search only for rep
142. ces introduced by Margaret Dayhoff These have been extremely widely used since the late 70s e GONNET these matrices were derived using almost the same procedure as the Dayhoff one above but are much more up to date and are based on a far larger data set They appear to be more sensitive than the Dayhoff series e D identity matrix which gives a score of 1 0 to two identical amino acids and a score of zero otherwise Iteration type specifies the iteration type to use During the iteration step each sequence is removed in turn and realigned It is kept if the resulting alignment is better than the one has been made before This process is repeated until the score converges or until the maximum number of iterations is reached Available values are e NONE specifies not to use iterations e TREE specifies to iterate at each step of the progressive alignment e ALIGNMENT specifies to iterate on the final alignment Max iterations maximum number of iterations 222 Unipro UGENE Manual Version 1 20 0 T Align with ClustalW Input file Output file Advanced options Gap opening penalty 15 00 Gap extension penalty L Weight matrix Iteration type NONE Max iterations 5 Out sequences order Protein gap parameters Gap separation distance E Hydrophilic gaps off No end gap separation penalty Residue specific gaps off The following parameters are only available for protein sequenc
143. ching Sequence Using HMM Profile HMM Search e Building HMM Model HMM3 Build e Searching Sequence Using HMM Profile HMM3 Search e Searching Sequence Against Sequence Database Phmmer Search e uMUSCLE e MUSCLE Aligning e Aligning Profile to Profile with MUSCLE e Aligning Sequences to Profile with MUSCLE ClustalW MAFFT T Coffee Bowtie e Bowtie Aligning Short Reads e Building Index for Bowtie e Bowtie 2 e Bowtie 2 Aligning Short Reads Building Index for Bowtie 2 e BWA e Aligning Short Reads with BWA e Building Index for BWA BWA SW e Aligning Short Reads with BWA SW 165 e Building Index for BWA SW BWA MEM e Aligning Short Reads with BWA MEM e Building Index for BWA MEM UGENE Genome Aligner e Aligning Short Reads with UGENE Genome Aligner e Building Index for UGENE Genome Aligner e Converting UGENE Assembly Database to SAM Format CAP3 SPAdes Weight Matrix e Searching JASPAR Database e Building New Matrix Primer3 e RTPCR Primer Design Spliced Alignment mRNA to genomic External Tools e Configuring External Tool Query Designer Plasmid Auto Annotation ClustalO Kalign Aligning Expert Discovery e Loading Sequences Mapping Sequences Markup Sequences Creating Signals Generating Signals Complex Signals Recognition on a Sequence Workflow Designer Unipro UGENE Manual Version 1 20 0 The Workflow Designer allows a molecular biologist to create and run complex computational workflow schemas even if he or she is not
144. cleotide Angle AA 7 6 CA 14 6 AC 10 9 CC 7 2 AG 8 8 CG 11 1 AT 12 5 CT 8 8 GA 8 2 TA 25 GC 8 9 TC 8 2 GG 7 2 TG 14 6 GT 10 9 TT 7 6 A minimum value is used when N characters is present in a dinucleotide CN NC GN NG NN 7 2 e AN NA TN NT 7 6 e Configuring Dialog Settings e Result Annotations Configuring Dialog Settings In the dialog you can setup the corresponding parameters Window size the number of bases in a window The window size should be greater than 2 The default value is 100 bp Window step the number of bases used to shift a window The Window step should be a positive integer The default value is 1 bp 168 Unipro UGENE Manual Version 1 20 0 Threshold the threshold value of the twist angle see above The default value is 13 7 You can remember the input values or restore the default values using the Remember Setting and the Restore Defaults buttons The annotations names and other parameters can be changed on the Output tab of the dialog T DNA Flexibility Search Settings Output w Save annotation s to Existing table NC_001363 features murine gb Create new table Use auto annotations table Y Annotaton parameters Group name lt auto gt Annotation name dna_flex Description Once the Search button has been pressed the annotations for the regions of the high DNA flexibility are created Result Annotations Each annotation has the following qualifiers e area_
145. ct Mycobacterium so 4 BAM SAM file import Mycobacterium sorte Task state description Running Running Running Running Finished Finished Importing reads m 84 Task progress 84 64 B45 84 100 100 Tasks 1 WP The Task name column of the Task View shows the tasks names Task state description shows the status of the active tasks Started Running Finished and so on The Task progress column shows the percentage of the tasks progress If you want to cancel a task click the red cross button in the Actions column for the task Log View The Log View shows the program log information To show hide the Log View click the Log button in the main UGENE window Unipro UGENE Manual Version 1 20 0 Log INFO 07 06 Converting assembly from Mycobacterium sorted bam to Mycobacterium sorted bam ugenedb started 5 INFO 07 07 Converting assembly from Mycobacterium sorted bam to Mycobacterium sorted bam ugenedb succesfully finished imported 967272 reads total time 50 s pack time 05 UNFO 07 07 BAMImporter task total time is 53 sec INFO 07 10 Converting assembly from Mycobacterium sorted bam to Mycobacterium sorted bam ugenedb started Log View INFO 07 11 Converting assembly from Mycobacterium sorted bam to Mycobacterium sorted bam ugenedb succesfully finished 2 imported 967272 reads total time 49 s pack time 0 s 7 INFO 07 11 BAMImporter task total time is 52 sec No active
146. ct file to export exported area and click on the Export button The task report will appear in the Notifications Zooming and Fonts To perform zoom operations use the corresponding buttons on the editor toolbar a ct e SZ By default the base characters are visible when zooming But for rather long sequences there is another zoom mode available In this mode the bases are not shown This allows viewing very large sequence regions up to 500 bp Zoom To Selection JJH _ gt mM 713 720 Ta Ta TEO TR m TRG Bd n gt At i tC sfi7o pro thi sf lez mags9 zmis ness wg i uns tnzz thos pzig nzie3 ENET DVZ viSZ5 viSSF 529173 529280 pime edIIREVCOMP 1i 2 z DAME ESEREVCOMP i E primer sd LIREWCOMP I 1 gt 3a You can zoom to the selected region by clicking the Zoom to selection button It is very convenient operation when the alignment size is rather large For example you can zoom out to some percentage select an interesting region and then zoom to the selection 122 Unipro UGENE Manual Version 1 20 0 You can change font by clicking the Change font button To reset zoom and font click the Reset zoom button Searching for Pattern You can search for a pattern inside an alignment Enter a query string in the edit box under the Sequence area E a jl jl Sti Find T Press the right arrow to search in the direction From left to right from top to bottom Pres
147. d in COlI aln out result sitecon Searching for TFBS with SITECON Task Name sitecon search Searches for transcription factor binding sites TFBS with SITECON and saves the regions found as annotations Parameters in semicolon separated list of input sequence files to search TFBS in String Required inmodel input SITECON profile s If several profiles have been supplied searches with all profiles one by one and outputs merged set of annotations for each input sequence String Required out output Genbank file String Required annotation name name of the annotated regions String Optional Default misc_feature min score recognition quality threshold The value must be between 60 and 100 Choosing too low threshold will lead to recognition of too many TFBS recognised with too low trustworthiness Choosing too high threshold may result in no TFBS recognised Number Optional Default 85 min err1 setting for filtering results minimal value of Error type I Number Optional Default 0 max err2 setting for filtering results maximum value of Error type II Number Optional Default 0 001 strand strands to search in Number Optional Default 0 The following values are available e 0 both strands e 1 direct strand e 1 complement strand Example 285 Unipro UGENE Manual Version 1 20 0 ugene sitecon search in input fa inmodel profile sitecon out res
148. d as an external tool into UGENE Open Tools Align to reference submenu of the main menu F T UGENE human_T1 s human_T1 UCSC April 2002 chr7 115977709 117855134 Q File Actions Settings Tools Window Help OG 6 B F a DNA assembly i Go a 7 H g i human _T1 UCSC April ir ikkini t Align short reads Hey HAMMER tools Build index Multiple alignment t 10k 20k 3 a i 20k Select the Align short reads item to align short reads to a DNA sequence using BWA SW Or select the Build index item to build an index for a DNA sequence which can be used to optimize aligning of short reads 232 e Aligning Short Reads with BWA SW e Building Index for BWA SW Aligning Short Reads with BWA SW Unipro UGENE Manual Version 1 20 0 When you select the Tools Align to reference Align short reads item in the main menu the Align Sequencing Reads dialog appears Set value of the Align short reads method parameter to BWA SW The dialog looks as follows M Align Sequencing Reads Alignment method Reference sequence Result file name Library Single end Short reads Index algorithm a Score for a match a Mismatch penalty b Gap open penalty q 5 Gap extention penalty 2 Band width w 50 SAM output Number of threads t Size of chunk of reads 3 1000000 Score threshold divided by match score T 30 F best z 1 Number of seeds to start
149. database much as BLASTP or FASTA would do The Phmmer search works essentially like the HMM3 search does except you provide a query sequence instead of a query profile HMM The database sequence must be selected in the Project View or there must be an active Sequence View window opened Select the query sequence in the Phmmer search dialog 217 Unipro UGENE Manual Version 1 20 0 ea aa Save annotation s to Existing table a COO Ey Create new table C Users yalgaer MyDocument gb Y Annotation parameters Group name auto gt Annotation name signal Length of sequences for MSV Gumbel mu fit Number of sequences for MSV Gumbel mu fit Length of sequences for Viterbi Gumbel mu fit Number of sequences for Viterbi Gumbel mu fit Length of sequences for Forward exp tail mu fit BH EH EH CH EH HH Number of sequences for Forward exp tail mu fit Tail mass for Forward exponential tail mu fit Eg The results are stored as sequence annotations in the Genbank file format 218 Unipro UGENE Manual Version 1 20 0 cy gil2156280 pir I38344 titin human TAA LTTE EEE EEE EEE sn Gn RR I y 1 200k 400k BOOK anok Tim 1 2m 1 4m 1 6m 1 8m ITI 2 2m LHEGHEYTEFRYSAENE 40 569 40575 40580 40505 40590 40595 406k 40605 40610 40615 4062 E g9 Annotations MyDocument_3 gb G8 signal 0 546 GH signal d761 9856 FH signal 10471 10549 GH signal 13660 13717 GH signal 36707 36782 GH signal 37397 37475
150. defined values by selecting the available preset 0 Find Tandems _2 e Tandem finder parameters Tandem preset Min period Max period Big Custom Region to process Region Whole sequence Min period Max period the minimum and maximum acceptable repeat length measured in base symbols Region to process specify the region to search in the whole sequence a custom region or the region of the current selection if any In the Save annotation s to group you can set up a file to store annotations It could be either an existing annotation table object a new annotation table or auto annotations table if it is available In the Annotation parameters group you can specify the name of the group and the name of the annotation If the group name is set to lt auto gt UGENE will use the group name as the name for the group You can use the characters in this field as a group name 189 Unipro UGENE Manual Version 1 20 0 separator to create subgroups If the annotation name is set to by tyoe UGENE will use the annotation type from the Annotation type t able as the name for the annotation Also you can add a description in the corresponding text field Advanced Advanced parameters Algorithm Suffix array optimized Minimum tandem size 9 Minimum repeat count x3 F Show overlapped tandems Additional search options can be found in the Advanced tab Algorithm the algorithm parameter allows to s
151. detailed information about different Options Panel tabs can be found in the following chapters e Options Panel in Sequence View e Information about Sequence e Search in Sequence e Highlighting Annotations e Options Panel in Assembly Browser e Navigation in Assembly Browser e Assembly Browser Settings e Assembly Statistic Adding and Removing Plugins A plugin is a dynamically loaded module that adds a new functionality to UGENE To manage plugins select the Settings Plugins main menu item The Plugin Viewer window will appear U New Project UGENE Plugin Viewer UJ Fie Actions Settings Tools Window Help Td Ot DAA Export Mame Description paumeto epokan d BALL A port of BALL Framework For m multiple DNA sequences a BioStruct3b viewer Plugin Biological 3D Structure Viewer D a Bowtie An ultrafast memory efficient sh oa CUDA Support Utility plugin For CUD4 enabled Description of a Chromaview Chromatograms visualization the selected oa Circularview Enables drawing of DNA sequen luain gt BNA Annotator This plugin contains routines to pugin t DNA Export Routines to export or align multi a DMA GraphPack Oni This plugin contains a set of gra a DNA Statistics Oni Provides statistical reports For 5 oa Dotplot On Build dotplot For sequences a External tool suppr On Runs other external tools he GORTY On GORIY protein secondary struct A HMM Rased on HMMER 2 3 2 package The list of plugins i
152. dpt ugenedb srfa srfasta UWI Read Unipro UGENE Manual Version 1 20 0 Write A multiple sequence alignments file format See also Alignment Editor An annotated protein sequence in format of the UniProtKB Swiss Prot database See also Sequence View A rich format for storing sequences and associated annotations produced by Vector NTI software See also Sequence View The VCF specifies the format of a text file used for storing gene sequence variations See also Assembly Browser Comment Stores a dotplot of a sequence See also Dotplot UGENE database files stores information for imported BAM or SAM files and can be used for converting this information into a SAM file See also mport BAM SAM File A multiple sequence alignments file format See also Alignment Editor Human readable format to store UGENE Workflo w Designer schemas See also Workflow Designer 293 UGENE Query uq Designer schema Workflow element for etc command line tool Other File Formats File format extension CSV html image formats bmp jpg png tiff svg etc pdf txt Unipro UGENE Manual Version 1 20 0 Human readable format to store UGENE Query Designer schemas See also Query Designer Format for storing workflow elements that can launch an external command line tool See also Workflow Designer Comment Exa
153. e accepts all annotations except the specified ones Boolean Optional complement complements the annotated regions if the corresponding annotation is located on the complement strand Boolean Optional extend left extends the resulting regions to the left for the specified number of base symbols Number Optional extend right extends the resulting regions to the right for the specified number of base symbols Number Optional gap length inserts a gap of the specified length between the merged annotations transl translates the annotated regions Boolean Optional Example 2 5 Unipro UGENE Manual Version 1 20 0 ugene extract sequence in sars gb out res fa annotation names gene Finding ORFs Task Name find orfs Searches for Open Reading Frames ORFs in nucleotide sequences and saves the regions found as annotations Parameters in semicolon separated list of input files String Required out output file with the annotations String Required name name of the annotated regions String Optional Default ORF min length ignores ORFs shorter than the specified length String Optional Default 100 require stop codon ignores boundary ORFs that last beyond the search region i e have no stop codon within the range Boolean Optional Default false require init codon allows ORFs starting with any codon other than terminator Boolean Optional Default
154. e Cirl A key sequence 56 Unipro UGENE Manual Version 1 20 0 To use the Sequence between selected annotations item select two annotations in the Annotations editor holding the Ctrl key at the same time eE 6 ENE L T E E E E E E E E o a E E E ae Mame value cy Me 001363 Features murine_copyz gb a Gl cbs 0 4 GQ misc_feature 0 2 ee E misc _feat note Goto position Ctrl G al tie _fea io Select sequence region Ctrl 4 poke Mew annotation Ctrl M by source 0 4 De oo Seguente region dd Sequence between selected annotations Analyze Sequence around selected annotations Export Align b H Edit sequence j H Remove Rulers ne fis 4nnokations highlighting And select the Select Sequence between selected annotations item in the context menu The Sequence around selected annotations item selects the selected annotations and the sequences between these annotations CY NC_001363 sequence DRT wa n 140335 1 IE 45k Jij We VEL Me J Ele 5 172 17 Sequence around selected annotations AS 1 1 GAGGAAACGGAGAGC PT TCAATACTGGCCETTCTCCTCITCT zo i TA RAGIT GCGAAAAAT a 1700 1705 1710 EZE TT TZ A 50 1755 CTCCTETGCCTGAUCRAGET AT SLAMAGAGGAGS CCTTTTTA R E 3 K 5s F YT r F ma rr Sequence between selected annotations Z fata Anmotat
155. e Options Panel Labels Show names E Show distances Align labels Aligning Labels To align a tree labels press the Align labels toolbar button or in the Tree settings Options Panel tab check the Align label item See the example of aligning labels below 159 Unipro UGENE Manual Version 1 20 0 oseeeeeee eee Podisma_sappaensis See LL soph ya a tir aE FEIET 2n Bicolorana_bocokr EFIE laa eel el el el al lalallala Montana_montana Metioptem japonica EF540831 Gampeockis sedakowl_EFD40528 eee Deracantha_deracantides EFI e ee Aytchia barano ooo eee eee Tettponia_ widissima soe ee eee Conovephalus_ discolor ooo e eee Conorephalus_ sp ait asta aa a aiaa iiaa F Conocephalus percaudata a A Mecopada elongata higaki_ J ane eai aa aa Mecopoda ebngata _Sumatra_ eee Mecopoda sp Malaysia_ al aa ll nll Phaneroptera_falcata Changing Labels Formatting To change formatting of a tree labels select the Labels Formatting toolbar button or the Tree settings Options Panel tab oo Font Helvetica z A Size 17 Attributes Here you can select color font size and attributes bold italic etc of the labels Note that when a clade has been selected the labels formatting settings are applied to the clade only Adjusting Branch Settings To adjust branch settings select the Branch Settings toolbar button the Branch Settings context menu item or the Tree settings Options Panel tab The following setti
156. e consensus sequences highlighted on the consensus sequence select the Show difference from reference item in the context menu of the Consensus Area or the Difference from reference item on the Assembly Browser Settingstab of the Options Panel 150 Unipro UGENE Manual Version 1 20 0 To export a Consensus Sequence right click on it in the Consensus Area and select the Export Export consensus item in the context menu For more information about consensus exporting see Exporting Consensus Exporting Exporting Reads Exporting Visible Reads Exporting Coverage Exporting Consensus Exporting Consensus Variations Exporting Assembly as Image Exporting Reads To export a read right click on it in the Reads Area and select the Export Current read item in the context menu The Export Reads dialog appears Export to file He format V Add to project Select a file to export the read to and the file format The read can be exported either to a FASTA or FASTQ file When the parameters are set click the Export button The read is exported to the file and if the Add to project check box has been checked it is added to the current project from where you can op en it Exporting Visible Reads To export all reads visible in the Reads Area select the Export Visible reads item in the Reads Area context menu The Export Reads dialog appears The dialog is described in the Exporting Read section Exporting Covera
157. e dialog will appear m Create New Project Projectname Mew Project Project folder Project file project 21 Unipro UGENE Manual Version 1 20 0 Here you need to specify the visual name for the project and the directory and file to store it After you click the Create button the Project View window is opened Creating Document To create a new sequence file from text select the File New document from text main menu item T UGENE Workflow Designer New workflow Cd New project New document from text ya Access remote database amp Connect to shared database Ctrl L A Search NCBI Genbank u Open Ctrl O0 fa Open as Ctrl Shift 0 The Create Document dialog appears m Create Document Faste data here E Custom settings Alphabet Standard DNA Skip unknown symbols Replace unknown symbols with SEQUENCE name Sequence E Save file immediately You can input the created sequence to the Paste data here field You can type or paste sequences in FASTA or text format The following Custom settings are available Alphabet here you can select the alphabet 22 Unipro UGENE Manual Version 1 20 0 Alphabet tang Skip unknown symbols Replace unknown symbols v Document location Document format The following alphabets are available Standard DNA Standard RNA Extended DNA Extended RNA Standard amino Extended amino Skip unknown symbo
158. e general syntax is the following ugene task task_name task_parameter value task_parameter value option value option value Here task_name task to execute it can be one of the predefined tasks or a task you have created task_parameter parameter of the specified task Some parameters of a task are required like in and out parameters of some tasks option one of the CLI options See the example below ugene align in COI aln out result aln log level details e CLI Options e CLI Predefined Tasks e Format Converting Sequences e Converting MSA Extracting Sequence Finding ORFs Finding Repeats Finding Pattern Using Smith Waterman Algorithm Adding Phred Quality Scores to Sequence Local BLAST Search Local BLAST Search Remote NCBI BLAST and CDD Requests Annotating Sequence with UQL Schema Building Profile HMM Using HMMER2 Searching HMM Signals Using HMMER2 Aligning with MUSCLE Aligning with ClustalW Aligning with ClustalO Aligning with Kalign Aligning with MAFFT Aligning with T Coffee Building PFM Searching for TFBS with PFM Building PWM Searching for TFBS with Weight Matrices Building Statistical Profile for SITECON Searching for TFBS with SITECON Fetching Sequence from Remote Database Gene by Gene Report Reverse Complement Converting Sequences Variants Calling e Generating DNA Sequence e Creating Custom CLI Tasks CLI Options help h lt option_name gt lt task_na
159. e homepage Bowtie is embedded as an external tool into UGENE Open Tools DNA Assembly submenu of the main menu 224 Unipro UGENE Manual Version 1 20 0 eer Window Help DNA assembly Align short reads Test runner Build index 4 SITECON t 3 Convert UGENE Assembly data base to SAM format Select the Align short reads item to align short reads to a DNA sequence using Bowtie Or select the Build index item to build an index for a DNA sequence which can be used to optimize aligning of the short reads to the sequence e Bowtie Aligning Short Reads e Building Index for Bowtie Bowtie Aligning Short Reads When you select the Tools DNA Assembly Align short reads item in the main menu the Align Short Reads dialog appears Set value of the Align short reads method parameter to Bowtie The dialog looks as follows I Align Sequencing Reads Alignment method Reference sequence Result file name Library F Prebuilt index SAM output Short reads Order Parameters Flags Vode E Colorspace clr Py Mag error o No Mag rounding nomaqround No forward orientation nofw No reverse complement orientation norc E Try as hard tryhard Best alignments best Seed seed s E All alignments all Seed length seedlen Maximum of backtracks maxbts Descriptors memory usage chunkmbs There are the following parameters Reference sequence DNA sequence to
160. e propinv a proportion of the sites are invariable 138 Unipro UGENE Manual Version 1 20 0 e invgamma a proportion of the sites are invariable while the rate for the remaining sites are drawn from a gamma distribution Gamma sets the number of rate categories for the gamma distribution You can select the following parameters for the MCMC analisys Chain length sets the number of cycles for the MCMC algorithm This should be a big number as you want the chain to first reach stationarity and then remain there for enough time to take lots of samples Subsampling frequency specifies how often the Markov chain is sampled You can sample the chain every cycle but this results in very large output files Burn in length determines the number of samples that will be discarded when convergence diagnostics are calculated Heated chains number of chains will be used in Metropolis coupling Set 1 to use usual MCMC analysis Heated chain temp the temperature parameter for heating the chains The higher the temperature the more likely the heated chains are to move between isolated peaks in the posterior distribution Random seed a seed for the random number generator Display tree in new window displays tree in new window Display tree with alignment editor displays tree with alignment editor Synchronize alignment with tree synchronize alignment and tree Save tree to file to save the built tree P
161. e read select the Copy current position to clipboard item in the Reads Area context menu Short Reads Vizualization There are various modes of reads highlighting and shadowing e Reads Highlighting e Reads Shadowing Reads Highlighting To apply a reads highlighting mode select it in the Reads highlighting menu of the Reads Area context menu or on the Assembly Browser Settings tab of the Options Panel The following modes are available e Nucleotide shows all nucleotides in different colors It is used by default 147 Unipro UGENE Manual Version 1 20 0 e Difference highlights gaps and nucleotides that differ from the reference sequence You should add a reference first for correct displaying of this highlighting a ee ee ee ates a a aie 9019 C240 e Strand direction highlights reads located on the direct strand in blue and reads on the complement strand in green e Paired reads highlights all paired reads in green Note that the information about the pair is shown in the hint M nnmonnan a4 4 4 Thm mt n m ano Te T rE n amp E 7 AT a Ga G a G i o it nnn n i i nanann 3j Er Er E e m m a e nnnnannn oe of amp amp D mn mo D Ei nomm mnm n gn Wea nnn sn re od ce oct cy ey n ri Eo Sint ee Dee oD a eo rE A ge ee ee r g m nnnm M BE c D D e Sir mn m Mi mn on nanman Ba n n mo slic im nn Reads Shadowing Various modes of c
162. e system and the path to it should be properly configured However there is no need in the additional configuration if you ve installed the UGENE Full Package as it already contains all the tools by default Otherwise if you ve installed the UGENE Standard Package you would need to configure an external tool in order to use it Note that in this case you can download the package with all the external tools from this page To learn how to configure an external tool read below e Configuring External Tool Configuring External Tool To configure an external tool 1 Make sure the tool is installed on your system 2 Seta path to the tool executable file in UGENE It can be set on the External Tools tab of the Application Settings dialog If the path hasn t been set for a tool UGENE menu items that launch the tool are displayed in italic For example on the image below a path for the ClustalW external tool has been set and paths for MAFFT and T Coffee has not 254 Unipro UGENE Manual Version 1 20 0 Edit LA a T T Al Align with MUSCLE 7 Tree M Align sequences to profile with MUSCLE J ii Statistics d M Align profile to profile with MUSCLE 7 View KC Align with Kalign 7 Export d Align with ClustalW 1 Advanced l i Ai with MAFT Consensus mode Align with FCoffee Query Designer The Query Designer allows a molecular biologist to analyze a nucleo
163. e temporary directory Building Index for UGENE Genome Aligner You can build an index to optimize short reads alignment using UGENE Genome Aligner To open the Build Index dialog select the Tools DNA assembly Build index item in the main menu Set value of the Align short reads method parameter to UGENE Genome Aligner The dialog looks as follows T Build Index Align short reads method Reference sequence Index file name Reference fragmentation 0 Mb Total memory usage 0 Mb System memory size 1536 Mb Soret Gree The parameters are the following Reference sequence DNA sequence to which short reads would be aligned to This parameter is required Index file name file to save index to This parameter is required Reference fragmentation this parameter influences the amount of parts the reference will be devided It is better to make it bigger but it influences the amount of memory used during the alignment 240 Unipro UGENE Manual Version 1 20 0 Total memory usage shows the total memory usage System memory size shows the total system memory size Converting UGENE Assembly Database to SAM Format To convert UGENE data base to SAM format click on the Tools gt DNA Assembly gt Convert UGENE assembly database to SAM format conte xt main menu item The following dialog will appear M Convert UGENE Assembly Database to SAM Format Assembly database Result SAM file Select assembl
164. ease the number of Gaps introduced Match scores reward and penalty for matching and mismatching bases Entrez query a BLAST search can be limited to the result of an Entrez query against the database chosen This restricts the search to a subset of entries from that database fitting the requirement of the Entrez query Examples are given below protease NOT hiv1 organism this will limit a BLAST search to all proteases except those in HIV 1 1000 2000 slen this limits the search to entries with lengths between 1000 to 2000 bases for nucleotide entries or 1000 to 2000 residues for protein entries Mus musculusforganism AND biomol_mrna properties this limits the search to mouse MRNA entries in the database For common organisms one can also select from the pulldown menu 10000 100000 mIlwt this is yet another example usage which limits the search to protein sequences with calculated molecular weight between 10 kD to 100 kD src specimen voucher properties this limits the search to entries that are annotated with a soecimen_voucher qualifier on the source feature 177 Unipro UGENE Manual Version 1 20 0 allffilter NOT enviromnental samplef filter NOT metagenomes orgn this excludes sequences from metagenome studies and uncultured sequences from anonymous environmental sample studies For help in constructing Entrez queries see the Entrez Help document Filters filters for regions o
165. eats that have an annotated region inside E Filter repeats that have an annotated region inside Nested repeats filter algorithm E Search for inverted repeats E Exdude tandems areas Estimated repeats count 0 The found repeats are saved and displayed as annotations to the DNA sequence human_T1 UCSC April 2002 chr7 115977709 1 Ok Name Value d cy Annotations MyDocument_1 gb a y repeat_unit 0 4 mM gt OF repeat_unit join 19912 19956 19990 20034 gt O repeat_unit join 63727 63804 65 54 65831 gt O repeat_unit Join 185499 185538 189058 189097 E gt O repeat_unit join 190533 190577 190640 190684 T 4 nr j Tandem Repeats Finding To find tandem repeats select the Analyze Find tandems context menu item in the Sequence View window In the opened dialog you can specify the tandem search parameters the region to search in and the result parameters 188 Unipro UGENE Manual Version 1 20 0 m Find Tandems Tandem finder parameters Tandem preset Min period Max period 1000000 n Region to process Regon Save annotation s to Existing table NC_014267 features NC_014267 1 gb Create new table Use auto annotations table Y Annotation parameters Group name lt auto gt Annotation name repeat unit Description The dialog parameters Tandem preset specify the tandem repeats parameters with pre
166. ed information DOESN T include any personal data Default settings this option resets the default settings on the next run Resources 38 Unipro UGENE Manual Version 1 20 0 T Application Settings General Resources Resources Network Application resources File Format Optimize for CPU count Directories Logging Alignment Color Scheme Imak External Tools Genome Aligner Workflow Designer OpenCLl Tasks memory limit On the Resources tab you can set resources that can be used by the application Optimize for CPU count Tasks memory limit and Threads limit Network 39 Application Settings General Resources Network File Format Directories Logging Alignment Color Sch External Tools Genome Aligner Workflow Designer OpenCL Unipro UGENE Manual Version 1 20 0 Network Preferred Web browser System default browser Custom browser Remote request settings Remote request timeout 60 sec Proxy Type Server C HTTP Use authentication with HTTP proxy Login Password Do not use proxy on following addresses separate line for each SSL settings Secure Socket protocol On the Network settings tab of the dialog you can specify Proxy server parameters select SSL settings and configure the Remote request timeout Preferred Web browser you can use either System default browser or specify some other browser File Format 40 Unipro UGENE Manual Version 1 20 0 T
167. elect either the Tools Cloning Digest into Fragments item or the Actions Cloning Digest into Fragments item in the main menu or the Clon ing Digest into Fragments item in the context menu The Digest Sequence into Fragments dialog appears m Digest Sequence into Fragments Restriction Sites Conserved Annotations Target Sequence human_Ti UCSC April 2002 chr 115977709 117855154 Available enzymes Selected enzymes Add gt Add All gt Remove Clear Selection Hint there are no available enzymes Use Analyze gt Find Restrictions Sites feature to find them On the Restriction Sites tab of the dialog you can see the name of the molecule the list of restriction enzymes found during the restriction analysis that can cut the molecule and the list of enzymes selected to perform the digestion To digest the sequence into fragments you should select at least one enzyme To move an enzyme to the Selected enzymes list click on it in the Available enzymes list and press the Add button Note that you can select several items in a list by holding the Ctrl key while clicking on the items To select all available enzymes press the Add All button To remove enzymes from the Selected enzymes list select them in the list and press the Remove button To remove all items from the Selected enzymes list press the Clear Selection button On the Conserved Annotations tab of the dialog you can select the annotations that must not be disrupted
168. elect the required parameters and press the Start button Building Index for BWA MEM To build BWA SW index select the Tools Align to reference Build Index item in the main menu The Build Index dialog will appears Set the Align short reads method parameter to BWA MEM The dialog looks as follows 23 Unipro UGENE Manual Version 1 20 0 T Build Index Align short reads method Reference sequence Index file name Index algorithm a E Colorspace c There are the following parameters Reference sequence DNA sequence to which short reads would be aligned to This parameter is required Index file name file to save index to This parameter is required Index algorithm a Algorithm for constructing BWA index Available options are It implements three different algorithms e is designed for short reads up to 200bp with low error rate lt 3 It does gapped global alignment w r t reads supports paired end reads and is one of the fastest short read alignment algorithms to date while also visiting suboptimal hits e pbwtsw is designed for long reads with more errors It performs heuristic Smith Waterman like alignment to find high scoring local hits Algorithm implemented in BWA SW On low error short queries BWA SW is slower and less accurate than the is algorithm but on long reads it is better e div does not work for long genomes Colorspace c the input is read in colorspace color
169. elect the search algorithm The default and a fast one is optimized suffix array algorithm Minimum tandem size the minimum tandem size sets the limit on minimum acceptable length of the tandem i e the minimum total repeats length of the searched tandem Minimum repeat count the minimum number of repeats of a searched tandem Show overlapped tandems check if the plugin should search for the overlapped tandems otherwise keep unchecked e Tandem Repeats Search Result Tandem Repeats Search Result An example of the search results for the micro satellite preset F CP001037 sequence O Rri i l 1 1m ei MNF TT 4m 5m Bm 7m 3 234 a22 oy i i i i i ii a M AA A A nh AA A MAA A MoA A MA aA AA A AR A AAA A AKA AR AA A A ok y 1 im 2m om ami om om Tm 6 234 322 H b of 5S K P E E R E R K S amp S L P amp amp P E Y C K C N R S H Q R E R E N H C H R H Q R D I Vs V I E A T R E R E K I I A I V T R E TGATATTGTAAGTGTAATCGAAGCCACCAGAGAGAGAGAGAAAATCATTGCCATCGTCACCAGAGA ire P ea gs Fal A g eo ell eee 4 Le Value E repeat unit join 7704727 7704727 7704728 7704728 7704729 7704729 7704730 7704730 7704731 7704731 7704732 7704732 77 O repeat unit join 1209587 1209588 1209589 1209590 1209591 1209592 1209593 1209594 1209595 1209596 1209597 1209593 E repeat unit join 2451669 2451670 2451671 2451672 2451673 2451674 2451675 2451676 2451677 2451678 2451679 2451580
170. election Note that when the Assembly Overview is in focus and you use either the zoom buttons on the toolbar the zoom items in the Actions main menu or a mouse wheel the Reads Area is resized appropriately The Assembly Overview can also be resized To zoom in the overview select either the Zoom in or the Zoom in 100x item in the Assembly Overview context menu You can scroll the resized overview by dragging the mouse while pressing down the mouse wheel To zoom out the overview select the Zoom out item in the context menu The Restore global overview item in the context menu restores the default overview size when the whole contig overview is shown Notice that the Assembly Overview shows the coordinates of the assembly areas visible in the Reads Area and in the Assembly Overview Reads Area coordinates 25 382 ta 26 031 650 bp Assembly Overview coordinates 23 738 to 27 954 4 216 bp To scroll the resized overview drag the mouse while pressing down the mouse wheel To learn about available hotkeys refer to Assembly Browser Hotkeys Ruler and Coverage Graph Description The Ruler shows the coordinates in the Reads Area When you move the mouse cursor in the Reads Area the coordinate of the selected location with the coverage of reads is shown on the ruler in dark red The Coverage Graph shows the exact coverage of the sequence at each position For example on the image below the coordinate is 9168 and the coverage of re
171. em in the context menu Ay s hur Open view Add to view i Unload selected documents Lock document for editing Add Import Export Edit Remove Export document Save selected documents The following dialog appears FEl Export Document Save to file Hie forma Compress file Add to project Here you may select the name of the output file in the Save to file field and optionally choose the format of the output file in the File format fi eld Use the Compress file checkbox to compress the file The Add to project checkbox checked by default adds the output file to the current project After choosing all parameters click the Export button Locked Documents The lock icon in the document element indicates that the document can t be modified Y 2 BH Sick icon MCE a 1CF7 chain 1 annotation Ys s LCF7 chain sequence a 1CF7 chain annotation UGENE does not allow modification of some formats that were created not by UGENE lf UGENE is able only to read a document see the Supported File Formats chapter you can export the document objects to a file To do it use the built in export utilities Also you can export the document objects of unlocked documents 25 Unipro UGENE Manual Version 1 20 0 Using Objects and Object Views The document always contains one or more objects An object is a structured biological data that can be visualized by different Object V
172. emble Genomes Assembly method Output directory Library Single end Properties Left reads Right reads Type Orientation Path Path Remove Base Options Dataset type Multi Cell j Running mode Error Correction and Assembly Number of threads t 8 l Memory limit GB m 250 gt k mer sizes k auto The following parameters are available Output directory SPAdes stores all output files in output directory which is set by the user Library to run SPAdes choose one of the following libraries e Single end e Paired end e Paired end Interplaced e Paired end Unpaired files e Sanger e PacBio Left reads file s with left reads Right reads file s with right reads For each dataset in the paired end libraries you can change type and orientation Datasest type dataset type Running mode running mode k mer sizes k k mer sizes Number of threads t number of threads Memory limit GB m memory limit Weight Matrix The Weight Matrix plugin is a tool for solving the problem of a sequence annotating As well as for the S TECON the main use case of the plugin is recognition of potential transcription factor binding sites on basis of the data about conservative conformational and physicochemical 244 Unipro UGENE Manual Version 1 20 0 properties revealed with the binding sites sets analysis The Weight Matrix contains a lot of position frequency matrices PFM s and
173. emove oo A New annotation Copy The parameters dialog will be re opened See description of the available parameters here Filtering Results It is possible to find features intersections and filter dotplot results Right click on the dotplot and select the Dotplot Filter results context menu item The following dialog will appear 113 Unipro UGENE Manual Version 1 20 0 H DotPict Dotplot parameters No Filtration Features Intersection Intersection Parameters Features Selection Feature Name 4 Sequence A CVU55 62 E gene E S0urce 4 Sequence Y NC_001365 F misc_feature Ae Select features and click OK button The filtered dotplot will appear Saving Dotplot as Image To save a doitplot as image right click on the dotplot and select the Dotplot Save Load Save as image context menu item Go to position Select sequence region New annotation Copy Palat The following dialog will appear 114 Unipro UGENE Manual Version 1 20 0 uU Export Image DotPlotExport settings Include area selection Include repeat selection Export to file File name C Users yalgaer untitled png Format Width 1830px gt Height 200px gt DPI g Available formats are png jpg omp jpeg ppm tif tiff xom and xpm Saving and Loading Dotplot To save a dotplot in a native format right click on the dotplot and select the Dotplot Save Load Save context
174. equence View Sequence View Components e Global Actions Sequence Toolbars Sequence Overview Sequence Zoom View Sequence Details View Information about Sequence Manipulating Sequence e Going To Position Toggling Views Exporting Sequence Image Zooming Sequence Creating New Ruler Selecting Amino Translation Showing and Hiding Translations Selecting Sequence Unipro UGENE Manual Version 1 20 0 e Copying Sequence e Search in Sequence e Load Patterns from File e Search Algorithm e Search in e Other Settings e Annotations Settings Editing Sequence Exporting Selected Sequence Region Exporting Sequence of Selected Annotations Locking and Synchronize Ranges of Several Sequences e Multiple Sequence Opening e Annotations Editor e db xref Qualifier e Automatic Annotations Highlighting e The comment Annotation e Manipulating Annotations e Creating Annotation e Selecting Annotations e Editing Annotation e Highlighting Annotations e Annotations Color e Annotations Visability e Show on Translation e Captions on Annotations Creating and Editing Qualifier Adding Column for Qualifier Copying Qualifier Text Finding Qualifier Deleting Annotations and Qualifiers Importing Annotations from CSV e Exporting Annotations e Sequence View Extensions e Circular Viewer e Circular View Settings e 3D Structure Viewer e Opening 3D Structure Viewer s Gnanging 3D Structure Appearance e Selecting Render Style Selecting Coloring Scheme Calcula
175. er criteria W Store only signals with different behaviour Minimal Complexity Maximal Complexity lt Back In the first dialog window extraction parameters see below are set Next windows are for setting operations which will be nodes of CS and choosing a folder for CS storing To see CS location in a sequence it is needed to pick sequences for representation with the popup menu of the sequence Then one can choose any CS and it will be shown as autoannotations on each represented sequence Moreover it is possible to observe few signals at once on the sequence for this user checks signals for group representation with the popup menu The same operation is used to choose signals for recognition Complex Signals Recognition on a Sequence After the CSs are automatically extracted they can be recognized on any sequence Such a set of sequences can be loaded as the control set For recognition some set of CSs is chosen each of the signals is applied to a sequence Then to a symbol of the sequence where CS is occurred log 1 P score is added where P is a value of conditional probability of the signal Score of the sequence is a total score of all its symbols The sequence is considered to be recognized when it has the selected CS and its total score is higher than the recognition bound Expert can choose the recognition bound using the training set Choosing of the recognition bound is performed in the corresponding dialog by clicki
176. erged annotations Number Optional Default 0 Example ugene query in input fa out result gb schema Repeat sWithORF ugql Building Profile HMM Using HMMER2 2 9 Unipro UGENE Manual Version 1 20 0 Task Name hmm2 build Builds a profile HMM using the HMMER2 tools Parameters in semicolon separated list of input multiple sequence alignment files String Required out output HMM file String Required name name of the profile HMM String Optional Default hmm_profile calibrate enables disables calibration Boolean Optional Default true seed random seed a non negative integer Number Optional Default 0 Example ugene hmm2 build in CBS sto out CBS hmm Searching HMM Signals Using HMMER2 Task Name hmm2 search Searches each input sequence for the significantly similar sequence that matches to all specified profile HMM using the HMMER2 tool Parameters seg semicolon separated list of the input sequence files String Required hmm semicolon separated list of the input HMM files String Required out output file with annotations String Required name name of the result annotations String Optional Default hmm_signal e val e value that can be used to exclude low probability hits from the result Number Optional Default 1e 1 score score based filtering which is an alternative to e value filtering to exclude low probability hits fro
177. es Gap separation distance tries to decrease the chances of gaps being too close to each other Gaps that are less than this distance apart are penalized more than other gaps This does not prevent close gaps it makes them less frequent promoting a block like appearance of the alignment Hydrophilic gaps off increases the chances of a gap within a run of hydrophilic amino acids No end gap separation penalty treats end gaps just like internal gaps to avoid gaps that are too close Residue specific gaps off amino acid specific gap penalties that reduce or increase the gap opening penalties at each position in the alignment or sequence For example positions that are rich in glycine are more likely to have an adjacent gap than positions that are rich in valine MAFFT Originally MAFFT is a multiple sequence alignment program for unix like operating systems However currently it is available for Mac OS X Linux and Windows It is used for both nucleotide and protein sequences MAFFT home page hitp mafft cbrc jp alignment software To make MAFFT available from UGENE e Install the MAFFT program on your system e Set the path to the MAFFT executable on the External tools tab of UGENE Application Settings dialog For example on Windows you need to specify the path to the mafft bat file To use MAFFT open a multiple sequence alignment file and select the Align with MAFFT item in the context menu or in the Actions main menu
178. es Show distances Align labels Hide font settings Font Helvetica Height Hide pen settings cr Line weight 1 pael Detailed information about tree setting see below e Selecting Tree Layout and View e Modifying Labels Appearance e Showing Hiding Labels e Aligning Labels e Changing Labels Formatting e Adjusting Branch Settings Selecting Tree Layout and View You can select one of the following tree layouts e Rectangular e Circular e Unrooted To do it press the Layout toolbar button and check the required item in the appeared menu or select it in the Tree settings Options Panel tab General ree yost Recan 7 Tree view Default See the example of the Circular layout 158 Unipro UGENE Manual Version 1 20 0 Also you can select one of the following tree view Default e Phylogram e Cladogram Modifying Labels Appearance From this paragraph you can learn how to show hide taxon and distance labels align them and change their formatting font color etc e Showing Hiding Labels e Aligning Labels e Changing Labels Formatting Showing Hiding Labels When you open a tree all the labels are shown by default To hide the taxon Sequence name labels select the Show Labels toolbar button or in the Tree settings Options Panel tab uncheck the Show Names item To hide the distance labels uncheck the Show Distances item To show the labels again check an appropriate item Labels settings in th
179. ew click the Project button in the main UGENE window Unipro UGENE Manual Version 1 20 0 UGENE File Actions Settings Tools Window Help ia gf St an Sa 4a aI Wy as chrM Jf 0 to 16 571 16 571 bp 4 if human_T1 fa s human_T1 U 8 737 C 146 Project view chrM sam bam as c T human_T1 s huma I Fi wW F F T W human_T1 s human_T1 UCSC April 2002 chr7 11 N E E E E M K E 5977709 11785 5 mE yN A TTGTCAGATTCACCAAAGCTTGAAATGAAGGAAAAAATG Pt tt tt tt tt 12 4 6 6 10 12 14 16 16 20 2 24 2H 2 30 2 H 3 AACAGTCTAAGTGCTTTCAACTTTACTTCCTTTTTTAC Q I You can also use the Alt 1 hotkey to show hide the Project View F S P F To create a new project refer to Creating New Project Note that if you have no project created when opening file with a sequence an alignment or any other biological data a new anonymous project is created automatically Task View The Task View shows active tasks for example algorithms computations To show hide the Task View click the Tasks button in the main UGENE window Task name a Loading documents Task View Load BAM info F Prepare assembly file to import E Convert BAM to UGENE database Myto UL 4 Okk GJ 3 Log Running task Loading documents The hotkey for showing hiding the Task View is Alt 2 a FT Opening view for document Mycobacterium sorted 4 F Adding document to proje
180. ext menu item and choose the annotation parameters in the Create Permanent Annotation dialog The comment Annotation General information about a file in GenBank or Vector NTI Sequence format stored in the COMMENT sections of the file is shown in UGENE in a special comment annotation in the Annotations Editor The information for example may include the name of the file author creation date and last modification date for the file and so on s 2xCyPET SxFLAG 6 features 2xCyPet 3xFLAG gt cs 0 3 a Q comment 0 1 4 comment Comment 1 4540 O1 This file is created by Vector NTI http warnw invitrogen com Author name Demo User Creation date Thu Dec 23 13 29 41 2010 Last modification date Thu Dec 23 15 29 41 2010 Object name 2xCyPET 3xFLAG 6xHIS pDONRP2R P3 p3 Owner P misc_feature 0 11 Manipulating Annotations 68 Unipro UGENE Manual Version 1 20 0 Creating Annotation Selecting Annotations Editing Annotation Highlighting Annotations e Annotations Color e Annotations Visability e Show on Translation e Captions on Annotations Creating and Editing Qualifier Adding Column for Qualifier Copying Qualifier Text Finding Qualifier Deleting Annotations and Qualifiers Importing Annotations from CSV Exporting Annotations Creating Annotation To create a new annotation for the active sequence press the Ctrl N key sequence select the New annotation toolbar button or use the Add New annotation or New anno
181. f SITECON provides a tool for recognition of potential binding sites for over 90 types of transcription factors Also UGENE version of SITECON provides a tool for recognition of potential binding sites basing site alignment proposed by user For the detailed method description see the original SITECON site Data about used context dependent conformational and physicochemical properties are available in the PROPERTY Database e SITECON Searching Transcription Factors Binding Sites e Types of SITECON Models e Eukaryotic e Prokaryotic e Building SITECON Model SITECON Searching Transcription Factors Binding Sites To search transcription factor binding sites in a DNA sequence select the Analyze Search TFBS with SITECON context menu item In the appeared search dialog you must select a file with TFBS profile The profiles supplied with UGENE are placed in the UGENE data sit econ_models folder After the profile is loaded the threshold filter is populated with values read from profile You can use the filter to remove low scoring regions from the result 205 Q SITECON Search Clear results Strands Both strands Direct strand 0 results found Save as annotations Unipro UGENE Manual Version 1 20 0 First type error Second type error The regions found by SITECON algorithm can be saved as annotations to the DNA sequence in the Genbank format Every S ITECON profile supplied with UGENE contains complete information abo
182. f low compositional complexity and repeat elements of the human s genome Masks for lookup table only this option masks only for purposes of constructing the lookup table used by BLAST so that no hits are found based upon low complexity sequence or repeats if repeat filter is checked Mask lower case letters with this option selected you can cut and paste a FASTA sequence in upper case characters and denote areas you would like filtered with lower case Filter by filters results by accession by definition of annotations or by id Select result by selects results by EValue or by score When the blastp search is selected in the general options the view of the Advanced options tab is the following Search Through a Remote Database General options Advanced options Word size i Gap costs Entrez query Matrix BLOSUM62 Service Filters Masks Low complexity filter Mask for lookup table only Human repeats filter Mask lower case letters Filter results Filter by Select result by E accession Evalue E def filter by definition of annotations Score E id As you can see there is no Match scores option but there are Matrix and Service options Matrix key element in evaluating the quality of a pair wise sequence alignment is the substitution matrix which assigns a score for aligning any possible pair of residues Service blastp service which needs to be performed plain psi or phi
183. f modern multicore processors and SSE instructions Out of the box support of modern GPUs using NVIDIA CUDA and ATI Stream Integrated solutions for Cell Broadband Engine Cooperation Can be used for education purposes in schools and universities Features to be included into the next release are initiated by users UGENE team is ready for collaboration in related projects both free and commercial Unipro UGENE Manual Version 1 20 0 Download and Installation UGENE is compatible with the three most common operating systems Windows Mac OS X and Linux It has some minimum system requirements If your system fits these requirements you re welcome to download UGENE from http ugene unipro ru download The program can be used and distributed under the terms of GPLv2 Follow these recommendation to choose which UGENE package to download Below you can also find links to the guides on UGENE installation on different operating systems System Requirements UGENE Packages Installation on Windows Installation on Mac OS X Installation on Linux e Native Installation on Ubuntu e Native Installation on Fedora System Requirements The system requirements for UGENE are these e Operating system 32 or 64 bit e Windows XP Windows Vista Windows 7 Windows 8 Using a zip package it is possible to use UGENE without administrative rights on Windows e Mac OS X 10 5 or later For older Mac OS X versions PowerPC 10 4 UGENE version 1 10 3 is a
184. fies the statistical significance threshold for reporting matches against database sequences Number Optional Default 10 hits maximum number of hits that will be shown Number Optional Default 10 name name of the result annotations If not set name will be specified with the cdd result or the blast result String Optional Default cdd or blast short optimizes search for short sequences Boolean Optional Default false blast output path to the file with the NCBI BLAST output only for the ncbi blastp and ncbi blastn databases Boolean Optional Default the file is not saved Example ugene remote request in seq fa db ncbi blastp out res gb Annotating Sequence with UQL Schema Task Name query Annotates a sequence in compliance with a UGENE Query Language UQL schema This allows to analyze a sequence using different algorithms at the same time imposing constraints on the positional relationship of the results To learn more about the UQL schemas read the Query Designer Manual Parameters in semicolon separated list of input sequence files String Required out output Genbank file with the annotations String Required schema UQL schema String Required merge if true merges regions of each result into a single annotation Boolean Optional Default false offset if merge is set to true specified left and right offsets for m
185. fox Opening data found using BioMart in UGENE Opening BioMart data in UGENE by ID Opening selected data in UGENE Unipro UGENE Manual Version 1 20 0 Unipro UGENE Manual Version 1 20 0 About Unipro Established in 1992 Unipro company has its headquarters located in Novosibirsk Akademgorodok the home of Siberian Branch of Russian Academy of Sciences The company s primary activity is IT outsourcing solutions To learn more about the company please visit the compa ny website Unipro UGENE Manual Version 1 20 0 About UGENE Unipro UGENE is a free cross platform genome analysis suite It is distributed under the terms of the GNU General Public License To learn more about UGENE visit UGENE website It works on Windows Mac OS X or Linux and requires only a few clicks to install Key Features User Interface High Performance Computing Cooperation Features Creating editing and annotating nucleic acid and protein sequences Search through online databases NCBI ENSEMBL PDB SWISS PROT UniProtKB Swiss Prot UniProtKB TrEMBL UniProt D AS Ensembl Human Genes DAS Multiple sequence alignment ClustalW ClustalO MUSCLE Kalign MAFFT T Coffee Online and local BLAST and BLAST search Restriction analysis with integrated REBASE restriction enzyme database Integrated Primer3 package for PCR primers design Search for direct inverted and tandem repeats in DNA sequences Constructing dotplots for nucleic acid sequences
186. g Labels e Aligning Labels e Changing Labels Formatting Adjusting Branch Settings e Zooming Tree Unipro UGENE Manual Version 1 20 0 Unipro UGENE Manual Version 1 20 0 e Working with Clade e Selecting Clade Collapsing Expanding Branches Swapping Siblings Zooming Clade Adjusting Clade Settings e Changing Root e Exporting Tree Image e Printing Tree e Extensions e Workflow Designer e DNA Annotator e DNA Flexibility e Configuring Dialog Settings e Result Annotations DNA Statistics DNA Generator ORF Marker Remote BLAST e Exporting BLAST Results to Alignment e Fetching Sequences from Remote Database e BLAST BLAST e Creating Database e Making Request to Database e Fetching Sequences from Local BLAST Database e Repeat Finder e Repeats Finding e Tandem Repeats Finding e Tandem Repeats Search Result e Restriction Analysis e Selecting Restriction Enzymes Using Custom File with Enzymes Filtering by Number of Hits Excluding Region Circular Molecule e Results e Molecular Cloning in silico e Digesting into Fragments e Creating Fragment e Constructing Molecule e Available Fragments Fragments of the New Molecule Changing Fragments Order in the New Molecule Removing Fragment from the New Molecule Editing Fragment Overhangs Reverse Complement a Fragment Other Constuction Options e Output e Creating PCR Product e In Silico PCR e Primers Details e Primer Library e Secondary Structure Prediction e SITECON e SITECON Searching
187. gb Fetching Sequence from Remote Database Task Name fetch sequence Fetches a sequence from a remote database The supported databases are accesed via alias Database Alias NCBI Genbank DNA genbank NCBI Genbank protein genbank protein Protein Data Bank pdb SwissProt swissprot Uniprot uniprot Parameters db database alias to read from String Required id semicolon separated list of resource IDs in the database String Required save dir directory to store sequence files loaded from the database String Optional Example ugene fetch sequence db PDB id 3INS 1CRN Gene by Gene Report Task Name gene by gene Suppose you have genomes and you want to characterize them One of the ways to do that is to build a table of what genes are in each genome and what are not there 1 Create a local BLAST db of your genome sequence contigs One db per one genome 2 Create a file with sequences of genes you what to explore This file will be the input file for the scheme 3 Setup location and name of BLAST db you created for the first genome 4 Setup output files report location and output file with annotated with BLAST sequence You might want to delete the Write Sequence element if you do not need output sequences 5 Run the scheme 5 Run the scheme on the same input and output files changing BLAST db for each genome that you have As the result you will get the report file With Yes and No
188. ge To export a coverage of the assembly select either the Export coverage item in the Consensus Area context menu The Export Coverage dialog appears 151 Unipro UGENE Manual Version 1 20 0 m Export the Assembly Coverage Export to Format Per base Compress the file Threshold i Additional options Export coverage value Export bases quantity Select a file threshold and format Histogram Per base or Bedgraph Where threshold is the minimum coverage value to export For Per base format the additional options are available Export coverage value or Export bases quantity or both of them When all the parameters are set click the Export button Exporting Consensus To export a consensus sequence of the assembly select either the Export consensus item in the Consensus Area context menu or the Expor t Consensus item in the Reads Area context menu The Export Consensus dialog appears T Export Consensus Export to file Fle format Sequence name chrM_consensus Consensus algoritm Keep gaps Add to project Select a file and the file format The consensus can be exported to a FASTA FASTQ GFF or GenBank file Modify if required the exported sequence name and choose the consensus algorithm The consensus is exported with gaps if the Keep gaps check box has been checked Also you can select the exporting region It can be either a Whole sequence a Visible region or a Custom region When all the par
189. ge keeps DNA sequences of several popular genomes such as human mouse drosophila melanogaster etc and hundreds of plasmid sequences Follow the instructions for accessing the storage 1 Use the menu File gt Connect to shared database or press the Ctrl L shortcut File Actions Settings Tools Window Help New project New document from text 6 Access remote database Connect to shared database Ctrl L Search NCBI Genbank Open Ctrl O Open as Ctrl Shitt O 0 es Recent Files b Recent Projects I Exit 2 Choose the predefined UGENE public database item and click the Connect button shared Databases Connections UGENE public database Disconnect 3 Browse the storage content 269 Unipro UGENE Manual Version 1 20 0 Project a Name filter Read only access 4 6 UGENE public database amp Recycle bin 4 genomes gt gt Arabidopsis thaliana TAIR 10 gt B C elegans ceb gt B Drosophila melanogaster dm3 d iS Escherichia coli str K 12 substr MG16 INO u Ys s NC_000913 a NC_000913 features gt gt Human hgl9 gt Human Immunodeficiency Virus HIV gt Mouse rm gt oS Mycobacterium tuberculosis NC_O00 gt gt Salmonella Enterica NC_016856 1 gt IE Vibrio cholerae NC_002505 gt O Yeast Saccharomyces cerevisiae sac gt 2 Zebrafish Danio rerio danRers gt gt plasmids The storage document i
190. gnment Working with Sequences List e Adding New Sequences Copying Sequences Renaming Sequences Sorting Sequences Shifting Sequences e Collapsing Rows Exporting in Alignment e Extracting Selected as MSA e Exporting Sequence from Alignment e Exporting Alignment as Image e Statistics Distance Matrix Grid Profile e Advanced Functions Building HMM Profile e Building Phylogenetic Tree PHYLIP Neighbor Joining MrBayes PhyML Maximum Likelihood e Assembly Browser e Import BAM SAM File Import ACE File Browsing and Zooming Assembly Opening Assembler Browser Window Assembly Browser Window Assembly Browser Window Components Reads Area Description Assembly Overview Description Ruler and Coverage Graph Description Go to Position in Assembly Using Bookmarks for Navigation in Assembly Data Getting Information About Read Short Reads Vizualization Reads Highlighting Reads Shadowing Associating Reference Sequence Associating Variations Consensus Sequence Exporting Exporting Reads Exporting Visible Reads Exporting Coverage Exporting Consensus Exporting Consensus Variations Exporting Assembly as Image Options Panel in Assembly Browser Navigation in Assembly Browser Assembly Statistics Assembly Browser Settings Assembly Browser Hotkeys Assembly Overview Hotkeys Reads Area Hotkeys e Phylogenetic Tree Viewer e Tree Settings Selecting Tree Layout and View Modifying Labels Appearance e Showing Hidin
191. gorithm implemented in BWA SW On low error short queries BWA SW is slower and less accurate than the s algorithm but on long reads it is better e div does not work for long genomes Number of threads t number of threads Min seed length k minimum seed length Matches shorter than NT will be missed The alignment speed is usually insensitive to this value unless it significantly deviates 20 Band width w band width Essentially gaps longer than NT will not be found Note that the maximum gap length is also affected by the scoring matrix and the hit length not solely determined by this option Dropoff d off diagonal X dropoff Z dropoff Stop extension when the difference between the best and the current extension score is above i A NT where i and j are the current positions of the query and reference respectively and A is the matching score Z dropoff is similar to BLAST s X dropoff except that it doesn t penalize gaps in one of the sequences in the alignment Z dropoff not only avoids unnecessary extension but also reduces poor alignments inside a long good alignment Internall seeds length r trigger re seeding for a MEM longer than minSeedLen FLOAT This is a key heuristic parameter for tuning the performance Larger value yields fewer seeds which leads to faster alignment speed but lower accuracy Skip seeds threshold c discard a MEM if it has more than INT occurence in the genome
192. h HMM profile 7 Save annotation s to Existing table eS NC_001363 features murine gb 5 Create new table Use auto annotations table Annotation parameters Group name auto gt Annotation type Misc Signal Annotation name hmm signal Description Expert options Filter results with E value greater then Filter results with Score lower than Number of sequences in dababase Algorithm The search results are stored as sequence annotations in the Genbank file format EF NC_000964 sequence Mae cz TE fi 2 Sle SN oo 2 342 436 2342 6k 2342 Tk ote OK a4 Ok 2o43k 2o43 1K 2343 2k 2543 3k 2 343 454 2 SAS TO tO ln NTT cease a E aka NER z34z Bk Z 342 80 lt Name Value cy Annotations MyDocument gb ew hmm_ signal 0 2 BB Amrm_ signal 2o42 756 2342992 bee Ewalue L4e 007 HMM model m3 0 kee Sire 22 8 Hi hmm_ signal 2257666 2257944 E gP NC_000964 features bsub gbk Q All HMM2 UGENE tools work only with files that contain a single HMM model HMM3 214 Unipro UGENE Manual Version 1 20 0 The HMM3 plugin is a toolkit based on the Sean Eddy s HMMERS package While working on this plugin we were guided by the following principles e Make the HMMERS tools accessible to a wider user audience by providing graphical interface for all supported utilities for most of the platforms e Be compatible with the original HMMER3 package e Create the high performance soluti
193. he Pho regulon in response to environmental Pi Member of the two component regulatory system phoQ phoP involved in adaptation to low Mg2 environments and the control of acid resistance genes PurR dimer controls several genes involved in purine nucleotide biosynthesis and its own synthesis Regulator capsule synthesis B Regulator capsule synthesis B Right origin binding protein Right origin binding protein SoxS is a dual transcriptional activator and participates in the removal of superoxide and nitric oxide and protection from organic solvents and antibiotics TorR response regulator Tryptophan trp transcriptional repressor Tyrosine repressor 209 Unipro UGENE Manual Version 1 20 0 To build a new SITECON model call the Tools gt S TECON gt Build new SITECON model from alignment main menu item The following dialog will appear 0 SITECON Build Input alignment nucleic Output model Options Window size Calibration random seed Calibration sequence length Weight algorithm Default status Here you need to select a nucleotide alignment and an output model Optionally you can change other parameters After that click on the Bu ild button Smith Waterman Search The Smith Waterman Search plugin adds a complete implementation of the Smith Waterman algorithm to UGENE To use the plugin open a nucleotide or protein sequence in the Sequence View and select the Analyze Find pattern Smith Waterman item in the co
194. hod Reference sequence Index file name E Colorspace There are the following parameters Reference sequence DNA sequence to which short reads would be aligned to This parameter is required Index file name a file to save the created index to This parameter is required Colorspace color the input is read in colorspace colors are encoded as characters A C G T A blue C green G orange T red Bowtie 2 Bowtie 2 is a popular ultrafast and memory efficient tool for aligning sequencing reads to long reference sequences Click this link to open Bowtie 2 homepage Bowtie 2 is embedded as an external tool into UGENE Open Tools Align to reference submenu of the main menu m UGENE human_T1 s human_T1 UCSC Apni 2002 chr7 115977709 117855134 a e B A amp DNA assembly Align to reference HMMER tools File Actions Settings Window Help Gol 4 7 id Align short reads Build index Multiple alignment BLAST Generate sequence SITECON Select the Align short reads item to align short reads to a DNA sequence Or select the Build index item to build an index for a DNA sequence which can be used to optimize aligning of the short reads to the sequence e Bowtie 2 Aligning Short Reads Building Index for Bowtie 2 Bowtie 2 Aligning Short Reads When you select the Too
195. hows the following information about the read e Read name e Location e Length e Cigar e Strand e Read sequence The operations in the Cigar parameter are described as follows e M Alignment match can be a sequence match or mismatch e Insertion to the reference Skipped when the read is aligned to the reference i e it is not shown in the Reads Area but is present in the read sequence e D Deletion from the reference Gaps are inserted to the read when the read is aligned to the reference For example From 5835 to 5886 Row 13 u Lenath 52 Deletion is shown as gaps Cigar 18M 3D3iM ele fer eee Abr Rew Strand direct i E E E e Ci TETE E A AL SCR 1 Read sequence CAAGGGAAGAGACCGATATACCCGCGCTGTCGAACTGCGATAAGATITI e N Skipped region from the reference Behaves as D but has a different biological meaning for mMRNA to genome alignment it represents an intron e S Soft clipping clipped sequences are present in the read sequence i e behaves as l e H Hard clipping clipped sequences are not present in the read sequence e P Padding silent deletion from padded reference e Exact match to the reference e x Reference sequence mismatch To copy the information about the read to the clipboard select the Copy read information to clipboard item in the Reads Area context menu Now you can paste it in any text editor To copy the current position of th
196. ialog will appear Resource ID NR 130109 Database NCBI GenBank DNA sequence Save to directory Output format gb Force download the appropriate sequence o x cae Hele Here you need to enter unique id of the biological object and choose a database The following databases are available NCBI Genbank DNA sequence NCBI protein sequence database ENSEMBL PDB SWISS PROT UniProtKB Swiss Prot UniProtKB TrEMBL Unique identifiers are different for various databases For example for NCBI GenBank such unique id could be Accession Number or NCBI GI number Optionally you can browse for a directory to save the fetched file to After you click the OK button UGENE downloads the biological object DNA sequence protein sequence 3d model etc and adds it to the current project If something goes wrong check the Log View it will help you to diagnose the problem UGENE Application Settings To open UGENE Application Settings dialog choose the Settings Preferences item in the main menu To open UGENE Application Settings dialog in Mac OS use the Unipro UGENE gt Preferences menu item The following settings are available General Resources Network File Format Directories Logging Alignment Color Scheme External Tools Settings Genome Aligner Workflow Designer Settings OpenCL 37 Unipro UGENE Manual Version 1 20 0 General T Application Settings ro nera General Resources Network Language of
197. iations 7 158 715 The variations will appear under the Consensus Sequence ate a Sh ee Variant nucleotides tT 15 380 C 251 15 390 To remove the association select the Remove track from the view item in the Variations Area context menu Consensus Sequence A consensus sequence can be found in the Consensus Area under a reference sequence It refers to the most common nucleotide at a particular position Reference sequence 7 J 340 q Consensus se quence i ee ee d eo ee ee me ee ee ee een ee eee ee ee ee ee en ee ee ee ee i oe E FE eee bee ee ee ee eS ee Se ee S ES E ES ee ee ee ES ee ee ee E ES pm E E EEE To choose a consensus algorithm select the Consensus algorihtm item either in the context menu of the Consensus Area in the context menu of the Reads Area or on the Assembly Browser Settings tab of the Options Panel The following algorithms are currently available e Default shows the most common nucleotide at each position When there is equal numbers of different nucleotides in a position the consensus sequence resulting nucleotide is selected randomly from these nucleotides e SAMtools uses an algorithm from the SAMtools Text Alignment Viewer to build the consensus sequence The algorithm takes into account quality values of reads and nucleotides and works with the extended nucleotide alphabet To leave only differences between the reference and th
198. ier e Automatic Annotations Highlighting e The comment Annotation db_xref Qualifier Some files in Genbank format contain the db_xref qualifier A value of this qualifier is a reference to a database 67 Unipro UGENE Manual Version 1 20 0 4 cy NC 004718 features sars gb gt W FuTR 0 1 gt W sur 0 1 a W cos 014 a E CDS CDS join 265 13398 13398 21485 codon_start 1 db_xret GE30124074 db xref GenelD 1 489680 gene orflab locus tag sarsl When you click on the value a web page is opened or a file is loaded specified in the reference The loaded file is added to the current project Automatic Annotations Highlighting Enabling the automatic annotations highlighting allows you to automatically calculate and highlight annotations on each nucleotide sequence opened Currently the following annotations types support the automatic highlighting e Open reading frames e Restriction sites e Plasmid features The corresponding groups of annotations found are stored in the Auto annotations object in the Annotations editor for example Auto annotations murine gb NC_001363 co g enzyme 8 0 H orf 0 27 To disable enable the automatic annotations calculations use the Automatic Annotations Highlighting menu button on the Sequence View too lbar a mA o Tr 3 6 Plasmid features ORFs w Restriction Sites To create a permanent annotation click on the Make auto annotations persistent cont
199. iew Percentage Identity Percentage Identity gray UGENE e Creating Custom Color Scheme Creating Custom Color Scheme To create custom color scheme use the Colors gt Custom schemes gt Create new color scheme context menu item The Application Settings di alog will appear Click on the Create color scheme button 119 Unipro UGENE Manual Version 1 20 0 m Application Settings General Alignment Color Scheme Resources Network Create and modify custom color schemes File Format Directory to save color scheme Logging a n Alignment Color Scheme External Tools Change color scheme Genome Aligner rl 1 Workflow Designer Delete The following dilaog will appear U Create Alignment Color Scheme New scheme name Custom color scheme Alphabet Nucleotide F Use extended mode Select the new scheme name alphabet and click on the Create button The next dialog will appear for nucleotide extended mode m Color Scheme Here you can select a color for each element Click on the element for it The new scheme will be created after clicking the OK button The new custom scheme will be available in the Colors gt Custom schemes context menu Highlighting Alignment 120 Unipro UGENE Manual Version 1 20 0 To apply an alignment highlighting mode select it in the Highlighting context menu Go to position Highlighting R No highlighting Edit Agreements Align Disagreements Tree Gaps Stat
200. iew Sequence Details View Information about Sequence Manipulating Sequence e Going To Position Toggling Views Exporting Sequence Image Zooming Sequence Creating New Ruler Selecting Amino Translation Showing and Hiding Translations Selecting Sequence Copying Sequence Search in Sequence e Load Patterns from File e Search Algorithm e Search in e Other Settings e Annotations Settings Editing Sequence Exporting Selected Sequence Region Exporting Sequence of Selected Annotations Locking and Synchronize Ranges of Several Sequences e Multiple Sequence Opening e Annotations Editor e db xref Qualifier e Automatic Annotations Highlighting e The comment Annotation e Manipulating Annotations e Creating Annotation e Selecting Annotations e Editing Annotation e Highlighting Annotations e Annotations Color e Annotations Visability e Show on Translation e Captions on Annotations Creating and Editing Qualifier Adding Column for Qualifier Copying Qualifier Text Finding Qualifier Deleting Annotations and Qualifiers Importing Annotations from CSV Exporting Annotations Sequence View Components The Sequence View is one of the major Object Views in UGENE aimed to visualize and edit DNA RNA or protein sequences along with their properties like annotations chromatograms 3D models statistical data etc For each file UGENE analyzes the file content and automatically opens the most appropriate view To activate the Sequence View open any fi
201. iews A single Object View can visualize one or several objects of different types For example a single view can show a sequence annotations for the sequence 3D model for the part of the sequence or its chromatogram simultaneously The type of an object is indicated by the symbol in the square brackets and the icon near the object SINS chain 2 sequence SINS chain 2 annotation SINS chain 3 sequence SINS chain 3 annotation SINS chain sequence SINS chain 4 annotation Below is the list of object tyoes supported by the current version of UGENE Object types Symbol 3d a as c i m s t tr Icon Description A 3D model Annotations for DNA sequence regions An assembly Chromatogram data A file with index information for a set of other usually large files A multiple sequence alignment A nucleic protein or raw sequence A plain text A phylogenetic tree You can edit names of particular objects such as sequence objects by selecting them in the Project View and then pressing F2 To be able to do so the document containing the target object must be unlocked To see the list of all available views for a given object select the object and activate the context menu inside the Project View window and select the Open view submenu 26 Unipro UGENE Manual Version 1 20 0 Project A F NC 001363 sequence Name Filter SZ k 500 ik a Objects ee muri e ho i
202. ignment or a file with several sequences to build the matrix from The parameter is mandatory Output file the resulting matrix will be saved in this file The parameter is mandatory Statistic type defines the way in which the statistics will be collected The Mononucleic option is basically good for small alignments and the Dinucleic option must give more appropriate results for big alignments Matrix type defines the type of the resulting matrix If the Frequency matrix option is selected then the frequency matrix will be created and saved into the resulting file If the Weight matrix option is selected then the intermediate frequency matrix will be created and then transformed into a weight matrix on basis of the selected Weight algorithm Then the weight matrix will be saved into the resulting file For some input files the colored Alignment Logo appears at the bottom of the dialog It gives the representation of the selected alignment 249 Unipro UGENE Manual Version 1 20 0 m Build Weight or Frequency Matrix Input file Output file statistic options Statistic type Mononudeic Matrix options Matrix type Frequency matrix Weight matrix Weight algorithm Berg and von Hippel Aall A The Alignment logo appears when e The input file format is pfm aln or it is a file with several sequences e The size of the input file is small enough To start the operation press the
203. ike HMMER OFR finding etc will use this code by default 54 Unipro UGENE Manual Version 1 20 0 Show codon table Translation frames 1 The Standard Genetic Code 2 The Vertebrate Mitochondrial Code 3 The Yeast Mitochondrial Code 4 The Mold Protozoan and Coelenterate Mitochondria and the Mycoplasma Code 5 The Invertebrate Mitochondrial Code 6 The Ciliate Dasycladacean and Hexamita Nuclear Code 9 The Echinoderm and Flatworm Mitochondrial Code 10 The Euplotid Nuclear Code 11 The Bacterial and Plant Plastid Code 12 The Alternative Yeast Nuclear Code 13 The Ascidian Mitochondrial Code 14 The Alternative Flatworm Mitochondrial Code 15 Blepharisma Nuclear Code 16 Chlorophycean Mitochondrial Code 21 Trematode Mitochondrial Code 22 Scenedesmus obliquus Mitochondrial Code 23 Thraustochytrium Mitochondrial Code The numbering of the genetic codes corresponds the NCBI Genbank database numbering Showing and Hiding Translations You can turn on off the direct and complement amino translations visualization in the Sequence details view using the Show complement strand and the Show amino translations toolbar buttons 140k Show translation buttons ko C D CU ee D D z 3 direct amino MWM T translations k L AATSCSEATAAAGAALGATSACTEC 5 1999270 195925 199930 199935 ITACSGCETATTTCTTCTACTGAG L AEE complement H I A amined translations I py L L H On the picture bel
204. ild HMMER3 profile item in the Actions main menu or in the context menu Learn more about the HMM tool in the documentation pages of the HMM2 and the HMM3 plugins Building Phylogenetic Tree To build a tree from an alignment either press the Build Tree button on the toolbar select the Tree Build Tree item in the alignment context menu or the Actions Tree Build Tree item in the main menu Fi Also you can use Tree Settings tab of the Options Panel 135 Unipro UGENE Manual Version 1 20 0 Tree Settings There are no displayed trees so settings are hidden Ca Open tree W Build tree Three methods for building phylogenetic trees are supported 1 The PHYLIP Neighbour Joining method The PHYLIP package implementation of the method is used under the hood 2 The MrBayes external tool Check MrBayes Web Site for more details 3 PhyML Maximum Likelihood method Check PhyML Maximum Likelihood Web Site for more details e PHYLIP Neighbor Joining e MrBayes e PhyML Maximum Likelihood PHYLIP Neighbor Joining The Building Phylogenetic Tree dialog for the PHYLIP Neighbour Joining method has the following view Build Phylogenetic Tree Distance matrix model E Gamma distributed rates across sites Coeffident of variation of substitution rate among sites 0 50 Transition transversion ratio 2 00 136 Unipro UGENE Manual Version 1 20 0 The following parameters are available Distance matrix model model
205. in semicolon separated list of input files String Required 281 Unipro UGENE Manual Version 1 20 0 out output file in the ClustalW format String Required Example ugene align kalign in COI aln out COI_aligned aln Aligning with MAFFT Task Name align mafft Multiple sequence alignment with MAFFT Q MAFFT is used as an external tool and must be installed on your system Parameters toolpath path to the MAFFT executable By default the path specified in the Application Settings is applied String Optional Default default tmpadir directory for temporary files String Optional in semicolon separated list of input files String Required out output file String Required format format of the output file String Required op penalty for opening a gap Number Optional ep penalty for extending a gap Number Optional maxiterate maximum number of cycles of iterative refinement Number Optional Example ugene align mafft in COI aln out COI_aligned aln Aligning with T Coffee Task Name align tcoffee Create alignment with T Coffee T Coffee is a collection of tools for computing evaluating and manipulating multiple alignments of DNA RNA Protein Sequences Q T Coffee is used as an external tool and must be installed on your system Parameters gap ext penalty Gap Extension Penalty Positive values give rewards to gaps and preven
206. in Project Options Panel Adding and Removing Plugins Searching NCBI Genbank Fetching Data from Remote Database UGENE Application Settings General Resources Network File Format Directories Logging Alignment Color Scheme External Tools Settings Genome Aligner Workflow Designer Settings OpenCL UGENE Terminology Project Storage for a set of data files and visualization options Document Unipro UGENE Manual Version 1 20 0 A single file can be stored on a local hard drive or be a remote web page Each document contains a set of objects Object A minimal and complete model of biological data For example a single sequence a set of annotations a multiple sequence alignment Task A process usually asynchronous that works in background For example some computations loading and writing files Plugin A dynamically loaded module that adds new functionality to UGENE Object View A graphical view for a single or a set of objects Project View A visual component used to manage active project Unipro UGENE Manual Version 1 20 0 Task View A visual component used to manage active tasks Log View A visual component used to show logs Notifications A visual component used to show notifications Generally it is used to open tasks reports Plugin Viewer A visual component used to manage plugins Sequence View An Object View aimed to visualize DNA RNA or protein sequences along with their properties like ann
207. ing dialog select the required parameters and click on the Export button 124 Alignment overview export settings Export simple overview Export graph overview Export to file File name ror Dispaly settings Unipro UGENE Manual Version 1 20 0 Graph type sets the graph type histogram line graph or area graph Orientation sets the orientation top to buttom or buttom to top Set color sets the gpaph color Calculation method sets the calculation method strict gaps clustal or highlighting To use these settings go to the corresponding context menu items of the alignment overview Working with Alignment This chapter explains how to work efficiently with the Alignment Editor You will learn how to modify an alignment remove gaps align sequences copy and paste regions add new sequences and extract subalignments as new alignments Undo Redo Framework Selecting Subalignment Moving Subalignment Editing Alignment Removing Selection Filling Selection with Gaps Replacing with Reverse Complement Replacing with Reverse Replacing with Complement Removing Columns of Gaps Removing All Gaps Saving Alignment Aligning Sequences Aligning Sequence to this Alignment Pairwise Alignment Working with Sequences List Adding New Sequences Copying Sequences Renaming Sequences Sorting Sequences Shifting Sequences Collapsing Rows Exporting in Alignment Extracting Selected as MSA Exporting Seque
208. ing will be disabled For long reads this option is typically ranged from 25 to 35 Max gap opens o maximum number of gap opens Index algorithm a algorithm for constructing BWA index It implements three different algorithms e is designed for short reads up to 200bp with low error rate lt 3 It does gapped global alignment w r t reads supports paired end reads and is one of the fastest short read alignment algorithms to date while also visiting suboptimal hits e bwtsw is designed for long reads with more errors It performs heuristic Smith Waterman like alignment to find high scoring local hits Algorithm implemented in BWA SW On low error short queries BWA SW is slower and less accurate than the s algorithm but on long reads it is better e div does not work for long genomes Best hits R proceed with suboptimal alignments if there are no more than specified number of equally best hits This option only affects paired end mapping Increasing this threshold helps to improve the pairing accuracy at the cost of speed especially for short reads 32bp Colorspace color the input is read in colorspace colors are encoded as characters A C G T A blue C green G orange T red Long scaled gap penalty for long deletion L long scaled gap penalty for long deletion Non iterative mode N disable iterative search All hits with no more than Max diff differences will be found This m
209. ion site will be found If it hasn t been checked the restriction site won t be found in this position Results When at least one enzyme has been selected and the OK button has been pressed in the dialog the auto annotating becomes enabled In the Annotations editor the Restriction Sites annotations can be found in the Auto annotations enzyme group The direct and complement cut site positions are visualized as triangles on an annotation in the Sequence details view TCGGCCBRBBSS TTrarc 9340 9345 9350 9355 ACCCCGGACGTCAAAATAG Molecular Cloning in silico This chapter describes a set of tools in UGENE to perform molecular cloning experiments in silico This allows you to digest a molecule into fragments create a fragment from a sequence region and ligate fragments into a new molecule e Digesting into Fragments e Creating Fragment e Constructing Molecule e Available Fragments Fragments of the New Molecule Changing Fragments Order in the New Molecule Removing Fragment from the New Molecule Editing Fragment Overhangs Reverse Complement a Fragment Other Constuction Options e Output e Creating PCR Product Digesting into Fragments Open a DNA molecule you want to cut into fragments Digestion into fragments is performed using restriction enzymes So before continuing make sure that the restriction analysis has been 192 Unipro UGENE Manual Version 1 20 0 performed Refer chapter Restriction Analysis for details S
210. ions L level C category YYYY or YY year MM month dd day hh hour mm minutes ss seconds zzz milliseconds By default logformat L hh mm license Shows license information lang language_code Specifies the language to use e g for the log output The following values are available e CS Czech 2 2 Unipro UGENE Manual Version 1 20 0 e EN English e RU Russian log color output If log output is enabled this option make it colored ERROR messages are displayed in red DETAILS messages are displayed in green TRACE messages are displayed in blue session db Session database is stored in the temporary file that is created for every UGENE run But it can supplied with the command line argument If the supplied file does not exest it will be created The session database file is removed after closing of UGENE For example ugene session db D session ugenedb version Shows version information tmp dir lt path_to_file gt Path to teporary folder ini file lt path_to_file gt Loads configuration from the specified ini file By default the UGENE ini file is used genome aligner UGENE Genome Aligner is an efficient and fast tool for short read alignment It has 2 work modes build index and align short reads default mode If there is no index available for reference sequence it will be built on the fly Usage ugene genome aligner option argument The fol
211. ipro UGENE Manual Version 1 20 0 Align short reads method Reference sequence Index file name index aos Ca E Colorspace c There are the following parameters Reference sequence DNA sequence to which short reads would be aligned to This parameter is required Index file name file to save index to This parameter is required Index algorithm a Algorithm for constructing BWA index Available options are It implements three different algorithms e is designed for short reads up to 200bp with low error rate lt 3 It does gapped global alignment w r t reads supports paired end reads and is one of the fastest short read alignment algorithms to date while also visiting suboptimal hits e bwtsw is designed for long reads with more errors It performs heuristic Smith Waterman like alignment to find high scoring local hits Algorithm implemented in BWA SW On low error short queries BWA SW is slower and less accurate than the is algorithm but on long reads it is better e div does not work for long genomes Colorspace color the input is read in colorspace colors are encoded as characters A C G T A blue C green G orange T red BWA MEM BWA is a fast light weighted tool that aligns relatively short reads to a reference sequence Click this link to open BWA homepage BWA ME M is generally recommended for high quality queries as it is faster and more accurate BWA MEM also has better pe
212. istics Conservation level View Transitions Export Transversions Advanced Use dots Set this sequence as reference Consensus mode APaddAANRngAgANOOPPrPAAnDSA Pe or on the Highlighting tab of the Options Panel Export The following modes are available e Agreements highlights symbols that coincide with the reference sequence e Disagreements highlights nucleotides that differ from the reference sequence e Gaps highlights gaps e Conservation level highlights conservation level of symbols in a multiple alignment gt or lt treshhold To select the conservation parameters use the Highlighting Options Panel tab 121 Unipro UGENE Manual Version 1 20 0 e Transitions highlights transitions e Transversions highlights transversions To use dots instead of symbols which are not highlighted check the Use dots checkbox in the Options Panel or use the Highlighting gt Use dots context menu item To select a reference sequence use the Set this sequence as reference context menu or Reference sequence field in the Highlighting tab of the Options Panel Also you can export highlighting with a help of the Export button in the Options Panel or by the Export gt Export highlighted context menu item The following dialog will appear Export Highlighted to File Export to file Exported area from OoOo to Indexing 1 based O based V Keep gaps V Dots instead not highlighted Sele
213. ith a help of the Cirl Spase the subalignment will be moved to the right by one column With a help of the Ctrl Backspase the subalignment will be returned to the first state Editing Alignment Select the Edit submenu in the Alignment Editor context menu Go to position Add Copy Colors Highlighting Edit Remove selection Align Fill selection with gaps Tree Replace selected rows with reverse complement Statistics Replace selected rows with reverse View Replace selected rows with complement Export Remove columns of gaps Shift Del Advanced Remove all gaps Set this sequence as reference Consensus mode The actions available from this menu are described below Removing Selection Filling Selection with Gaps Replacing with Reverse Complement Replacing with Reverse Replacing with Complement Removing Columns of Gaps 126 Unipro UGENE Manual Version 1 20 0 e Removing All Gaps Removing Selection To remove a subalignment select it and choose the Edit Remove selection item in the context menu or press the Delete key For Mac OS use the Fn Delete key instead of the Delete key Filling Selection with Gaps Select a region in the alignment and choose the Edit Fill selection with gaps item in the context menu or press the Spacebar The region is filled with gaps shifting the subalignment from the region to the right Replacing with Reverse Complement To replace sequence s in the alignment with re
214. ither in the 3D Structure Viewer context menu or in the Display menu on the toolbar to do it To stop the spinning uncheck the Spin item Selecting Sequence Region When you are selecting a region of a sequence e g in the Sequence zoom view the corresponding region on the 3D structure is being highlighted while the rest regions of the 3D structure are being shaded To configure the color of a region selected open the Settings dialog press the Settings item in the 3D Structure Viewer context menu or in the Display menu on the toolbar to do it press the Set selection color button and select a color in the dialog appeared To adjust the shading drag the Unselected regions shading slider in the Settings dialog 94 Unipro UGENE Manual Version 1 20 0 i 3D Structure Viewer Active view 1 2ZNL 2 Display oo CY 22NL chain 1 sequence amino rA ie Selecting Models to Display When a molecular structure contains multiple models e g NMR ensembles of models the Models item appears in the 3D Structure Viewer context menu and in the Display menu on the toolbar Render Style Coloring Scheme Molecular Surface Render Style Molecular Surface Models Spin Settings Export Image Structural Alignment Close The dialog will appear 95 Unipro UGENE Manual Version 1 20 0 of Select Models To show all the models check the All item To sho
215. itions are available e inside all labels are inside of the annotations e outside all labels are outside of the annotations e inside outside if the label can fit the annotation and it is not auto annotation it s located inside Otherwise outside e none no labels at all 3D Structure Viewer The 3D Structure Viewer is intended for visualization of 3D structures of biological molecules Using the 3D Structure Viewer you can work with data from the Protein Data Bank PDB a repository for the 3D structural data of large biological molecules such as proteins and nucleic acids maintained by the Worlwide Protein Data Bank wwPDB You can work as well with data from the NCBI Molecular Modeling DataBase MMDB also known as Entrez Structure a database of experimentally determined structures obtained from the RCSB Protein Data Bank Find the description of the 3D Structure Viewer features below e Opening 3D Structure Viewer e Changing 3D Structure Appearance e Selecting Render Style Selecting Coloring Scheme Calculating Molecular Surface Selecting Background Color Selecting Detail Level 89 Unipro UGENE Manual Version 1 20 0 e Enabling Anaglyph View Moving Zooming and Spinning 3D Structure Selecting Sequence Region Selecting Models to Display Structural Alignment Exporting 3D Structure Image Working with Several 3D Structures Views Opening 3D Structure Viewer The 3D Structure Viewer is opened autom
216. je new_qualifier To edit a qualifier select the qualifier and press the F4 key or use the Edit qualifier context menu item Name Value gP NC 001363 Features murine_copy gb a GM cbs 0 4 em g misc_Feature 0 2 O misc Feature 2 5 ae re eet I Gi E mi Copy qualifier mote value 45 H A sourd 2 Add note column Edit qualifier F4 amp Go to position Chrl 6 3 Select sequence region Ctrl 4 T 2 Tasks 4 New annotation Ctrl M Copy Select Add Analyze d Align Adding Column for Qualifier It is possible to add a column with the qualifier values to the Annotations editor To add the column select the Add the qualifier name column qualifier context menu item Copying Qualifier Text Use the Copy qualifier the qualifier name text qualifier context menu item to copy the qualifier value Finding Qualifier To find a qualifier select annotation s or group s of annotations and use the Find qualifier context menu 74 Unipro UGENE Manual Version 1 20 0 a Name Value 9 Auto annotations CVU55 62 gb CVUS5 62 F cy CVU55 62 features CVU55 62 gb F cne SE GB Find qualifier Aat Invert annotation selection db xref Rename ttem F2 gene ai dud Goto position Ctrl G protein_id Select sequence region Ctrl A transl_table 4 New annotation Ctrl N translation gt O CDS Copy i
217. join the sequences you can also select the Gap size The gap of the specified size will be inserted between the joined sequences After you press the Next button the dialog to configure the dotplot parameters will appear H DotPlot Dotplot parameters X axis SEQUENCE Y axis se quence Search for direct repeats E Search for inverted repeats F Custom algorithm Minimum repeat length Repeats identity The following parameters are available X axis sequence the sequence for the X dotplot axis Y axis sequence the sequence for the Y dotplot axis If there are several sequences in the specified the first or the second file and you haven t selected to join the sequences in the previous dialog then you can select a sequence in these fields If you have selected to Join all sequences found in the file then you can t select a separate sequence from the file the joined Sequence can be selected instead Search direct repeats check this option to search for direct repeats in the specified sequences You can also select the color with which the repeats will be displayed in the picture The default button sets the default color Search inverted repeats check this option to search for inverted repeats in the specified sequences You can also select the color with which the repeats will be displayed in the picture The default button sets the default color Custom algorithm optionally you can select an algo
218. l Bate ys STE penis HR angle ermine Pag a Sod aoe 1 oo sss ae m Geo I wa Ope ia 4 see oa p em tee hk of Bago Pp E ACE i ho n oes oo oo WOM aaa Ee Pe nae ye Geeta SP pee ce ee ee a a err Hee hie aec eee E oo a5 a Epa Te a ma E a cere a a bee 2 a Eis G 5 ui a x Oa I A ui Ci Ee oaa an a e aa meme a a Eoen ea Mn ana aaan Saak P mio m mtn Cee ee SCS Specs Go a an x Oo oe Coe od ct Raia I AN m 8 pega dg a a nn n F oy fe 0s oo Oe Ce ee ee eae hog an r Ser m s a Gea Uae Ce ate Geaao Po n Pi Go An a a ate a 1 mal at re sl RMI a lie al J oe hg a on eee tn Bh erie ie a a a a ete ta ee eee Tr an nat San eo a 50k 100k 110k 120k 1 10k 20K 30k 40k 0k 60k FOK GOK 90k 100k 110k 120k 140 426 W L L P E WF N D P R F S E L W E I TGGTACTATTACCCGAGGTTAACGATCCTAGGTTTAGTAAGTTOGTCGAAATT ett S S S S S S S S ST CO ES S S rs S FO S OO SE S S S 12 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 53 109 Unipro UGENE Manual Version 1 20 0 It is a two dimentional plot consisted of dots Each dot on the plot corresponds to a matched base symbol at the x position of the horizontal sequence and the y position of the vertical sequence Visible diagonal lines indicate matches between sequences in the given particular region See also e Interpreting Dotplot Identifying Matches Mutations Invertions etc
219. le menu it can be found either in the 3D Structure Viewer context menu or in the the Display menu on the toolbar Ball and Stick Worms Selecting Coloring Scheme You can select one of the following coloring schemes e Chemical Elements e Molecular Chains e Secondary Structure e Simple colors To change the coloring scheme open the Coloring Scheme menu available in the context menu and in the Display menu on the toolbar 91 Unipro UGENE Manual Version 1 20 0 Chemical elements Molecular chains Secondary structure Simple colors Calculating Molecular Surface To calculate the molecular surface of a molecule select the Molecular Surface item in the 3D Structure Viewer context menu or in the Display menu on the toolbar and check one of the following items e SAS solvent accessible surface e SES solvent excluded surface e vdWS van der Waals surface To remove the molecular surface that has already been calculated select the Off item You can also select the Molecular Surface Render Style to modify the calculated molecular surface appearance e Convex Map e Dots 92 Unipro UGENE Manual Version 1 20 0 SAS solvent accessible surface SES solvent excluded surface vdW S van der Waals surface vdW S with dots Selecting Background Color To change the background color open the Settings dialog choose the Settings item in the 3D Structure Viewer context menu or in the Displa y menu on the t
220. le with at least one sequence For example you can use the UGENE data samples EMBL AF 177 870 emb file provided with UGENE After opening the file in UGENE the Sequence View window appears 46 Unipro UGENE Manual Version 1 20 0 UGENE murine s NC_001363 File Actions Settings Tools Window Help M NC_001363 dna aK 5245 589 3 E Type Value Misc Feature 2 090 Misc Feature 5245 5833 gt Source w 1 No active tasks g A After the view is opened you can see a set of new buttons in the toolbar area The actions provided by these buttons are available for all sequences opened in the view In the picture below these buttons are pointed by the Global actions arrow Below the toolbar there is an area for a single or several sequences For each sequence a smaller toolbar with actions for the sequence and the following areas are available UGENE murine s NC_001363 File Actions Settings Tools Window Help 4k 1 2k 1 4k 1 6k T D P a H Details View T E vV 3 W c 1 CCCACCCACCACCCACCCACCACCCCCACCTAACCTCCCCACCAACTTATCTCTG G ma CCCTGGTGGCTGGGTGGTGGCCCTCCATTOUGACCOG Bo GTTGAAT PARAR TT 1 i ik A 6 0 66 T gma ay Ei G a w misc feature 0 2 e E misc_feature Misc Feature 2 590 e E misc_feature Misc Feature 5245 5833 No active tasks W EJ 47 Unipro UGENE Manual Version 1 20 0 An example of the Sequence View with several sequences T U
221. lignment Color Scheme Create and modify custom color schemes Directory to save color scheme mm Change color scheme Delete On the Alignment Color Scheme tab you can create change and delete custom color schemes 43 Unipro UGENE Manual Version 1 20 0 External Tools Settings Here you can set the paths to the external tools executable files Application Settings General Resources Network File Format SORT SINT ELET eas Legging Alignment Color Scheme External Tools Name Genome Aligner i es OpenCL aa Workflow Designer E Bowtie 2 index i E Bowtie 2 build in E Bowtie 2 aligner 4 B Bowtie E Bowtie build ind E Bowtie aligner Supported tools MW came pm tok ye ad i C work ext_tools_win_64 bit blast 2 2 28 bin blastdbcmd exe C work ext_tools_win_64 bit blast 2 2 28 bin rpsblast exe C work ext_tools_win_64 bit blast 2 2 28 bin tblast exe C work ext_tools_win_64 bit blast 2 2 28 bin tblastn exe C work ext_tools_win_64 bit blast 2 2 28 bin blastx exe m Additional information Select an external tool to view more information about it Genome Aligner Use this tab to configure the Genome Aligner settings Application Settings General Genome Aligner Resources Network Directories File Format Directory for built indexes Logging Alignment Color Scheme External Tools Genome Aligner OpenCl Workflow Designer Workflow Designer Settings
222. lished connection can be terminated by pressing the Delete button The same effect is produced by removing the database document item from Project View Adding Data to the Database To add data to the database use the Add gt mport to the database context menu item of the database in the project tree view Also you can drag n drop it to a shared database folder T UGENE CVU55762 s CVU55 ra File Actions Settings Tools Window Help a amp Si A Ga Ge Ga Bn Ga Fe Gob Add 4i Import to the database Edit Add folder Export Import ay Remove selected items gt gt GFF gt iS Genbank gt MMDB gt MSF The following dialog will appear 265 Unipro UGENE Manual Version 1 20 0 ltem to import General options Remove Here you can add to the database files folders or other objects from the current Project View To do this use corresponding buttons After specifying your data click on the mport button The data will be imported and appear in the database data tree Also you can change import settings To do this click on the General options button The following dialog will appear Database Import Default Options Destination folder Files and folders options Process directories recursively Keep folders structure Create a subfolder for the top level folder Create a subfolder for each file E Import unrecognized files Multi sequence files import policy
223. location to select a file format and an amino translation to export whole alignment or selected rows and optionally add the created document to the current project Export Sequences Associated with Annotation In UGENE you can export a sequence associated with an annotation To do it select the annotation in the Project View window and click the Export Import Export corresponding sequence context menu item 31 Unipro UGENE Manual Version 1 20 0 d it human_Tl fa s human_Tl UCSC April 2002 4 if murine gb s NC_001363 IE W a NC 00r H Open view b Add to wiew P H P kti A a Edit b ppotisupodt Export annotations Remove selected items Del D ae Open containing folder Import annotations from CSV file The Export Selected Sequences dialog will appear T Export Selected Sequences Export to file File format to use Export with annotations Add document to the project E Use custom sequence name murine Converton optons Save direct strand O Save complement strand O Save both strands Save both strands Save all amino frames Use custom translation table 1 The Standard Genetic Code Merge options Save as separate sequences Merge sequences Add gap symbols between sequences Here you can select the location of the result file and a sequence file format You can choose to add newly created document to the current project and use cust
224. lowing options are available build index Use this flag to only build index for reference sequence reference Path to reference genome sequence short reads Path to short reads data in FASTA or FASTQ format index Path to prebuilt index base file name or with idx extension If not set index is searched in system temporary directory If build index option is applied index will be saved to specified path result Path to output alignment in UGENEDB or SAM format see sam memsize Memory size in Mbs reserved for short reads The bigger value the faster algorithm works Default value depends on available system memory ref size Index fragmentation size in Mbs Small fragments better fit into RAM allowing to load more short reads Default value is 10 n mis Absolute amount of allowed mismatches per every short read mutually exclusive with pt mis Default value is 0 pt mis Percentage amount of allowed mismatches per every short read mutually exclusive with n mis Default value is 0 rev comp Use both the read and its reverse complement during the aligning best Report only about best alignments in terms of mismatches omit size Omit reads with qualities lower than the specified value Reads which have no qualities are not omitted Default value is 0 sam Output aligned reads in SAM format Default value is false For example 273 Unipro UGENE Manual Version 1 20 0 Build index for reference seq
225. ls Replace unknown symbols with you can select either to skip unknown input symbols or to replace them with the specified symbol Document location location of the created document Document format format of the created document Currently available formats are FASTA and Genbank Sequence name name of the sequence in the created document Save file immediately check this option if you want to save the document immediately after the Create button is pressed The created document will be added to the current project and opened in the Sequence View Opening Document UGENE stores information about documents you are working with in a project Once a document has been opened the information about it is saved in the current project e Opening for the First Time e Advanced Dialog Options e Opening Document Present in Project e Opening Several Documents Opening for the First Time To open a document that is not yet presented in the current project use either an advanced Open dialog a simple open file dialog or just drag the document to the UGENE window UGENE automatically detects the format of the document but if you use the advanced dialog you can choose the format manually To open the advanced dialog select one of the following e Add Existing document item in the Project View context menu e File Open As item in the main menu To simply open the document select one of the following e Open item in the main tool
226. ls Align to reference Align short reads item in the main menu the Align Sequencing Reads dialog appears Set value of the Align short reads method parameter to Bowtie 2 The dialog looks as follows 221 Unipro UGENE Manual Version 1 20 0 m Align Sequencing Reads slgrment meth Reference sequence Result fle name Library Prebuilt index SAM output Short reads Parameters Flags Mode No unpaired alignments no mixed Number of mismatches No discordant alignments no discordant C Seed length No forward orientation nofw C Add columns to allow gaps dpad T No reverse complement orientation norc E Disallow gaps gbar E No overlapping mates no overlap Seed seed No mates containing one another no contain There are the following parameters Reference sequence DNA sequence to align short reads to This parameter is required Result file name file in SAM format to write the result of the alignment into This parameter is required Library single end or paired end reads Prebuilt index check this box to use an index file instead of a source reference sequence The index is a set of 6 files with suffixes 1 ebwt 2 ebwt 3 ebwt 4 ebwt rev 1 ebwt and rev 2 ebwt The index is created during the alignment Also you can build it manually SAM output always save the output file in the SAM format the option is disabled for Bowtie Short reads
227. lyze Align Align sequence to mRNA Cloning a Align selected sequence regions Export Align selected sequence regions amino acids Edit sequence Align selected annotations Remove Align selected annotations amino acids Rulers Statistics In the list of sequences select the corresponding mRNA sequence and click OK The following dialog will appear U Save result to annotation g9 NC_001363 features murine gb Create new table Use auto annotations table Here you can set up a file to store annotations It could be either an existing annotation table object or a new annotation table or auto annotations table if it is possible Also you can modify the group name parameter and add a description The resulting alignment will be saved as an annotation with the corresponding name 253 Unipro UGENE Manual Version 1 20 0 T UGENE dna s dna wy File Actions Settings Tools Window Help Cd S 4 a Ga Sn ay Ga Fa Sa T i dna dna a r Tr 100 200 300 400 sbo 600 70 i 4 2 1 4k 15k 1608 142 bp 142 273 223 bp 495 263 bp M M 4 Name Value a ay Annotations MyDocument gb 4D exon 0 1 ey Aute annotations dna tet dna 4 mT External Tools The External Tools plugin allows one to launch an external tool from UGENE To use an external tool from UGENE the tool needs to be installed on th
228. m the result Number Optional Default 1000000000 Example ugene hmm2 search segq CBS_seq fa hmm CBS hmm out CBS_hmm gb Aligning with MUSCLE Task Name align Performs multiple sequence alignment with MUSCLE algorithm and saves the resulting alignment to file Source data can be of any format containing sequences or alignments Parameters in Input alignment Url datasets max iterations Maximum number of iterations using 2 by default Number mode Selector of preset configurations that give you the choice of optimizing accuracy speed or some compromise between the two The default favors accuracy using MUSCLE default by default Number range Whole alignment or column range e g 1 100 using Whole alignment by default String stable Do not rearrange aligned sequences using True by default Boolean format Document format of output alignment using clustal by default String out Output alignment String Example ugene align in test aln out test_out aln format clustal 280 Unipro UGENE Manual Version 1 20 0 Aligning with ClustalW Task Name align clustalw Multiple sequence alignment with ClustalW Q ClustalW is used as an external tool and must be installed on your system Parameters toolpath path to the ClustalW executable By default the path specified in the Application Settings is applied String Optional Default default
229. manually create Complex Signal one can use the context menu of the Complex signals item rr Complex signals New folder New signal Select all signals Deselect all signals Sort Also grouping folders are provided for convenience Under definition of CS it is represented as a hierarchical tree in which the operations are nodes and markups items or words are leafs When CS is created and selected its structure can be changed and parameters can be viewed in the parameters area The available types of nodes are the distance operation binary the repetition operation the interval operation the markup items and words CS is full determined when all its leafs have terminal symbols words or markup items Generating Signals Using the training set positive and negative set markups the system can construct a structure of a regulatory region as Complex Signal The extracting wizard is launched by the Extract signals button on the toolbar 260 Unipro UGENE Manual Version 1 20 0 T UGENE Expert Discovery File Actions Settings Tools Window Help 8368 86S A amp w The following dialog will appear m Extractor Parameters Setup Setup algorithm parameters This wizard will help you automaticaly extract complex signals from sequences Please fill in selection parameters Condition probability level Coverage bound Fisher criteria level Check minimization of Fish
230. me gt Shows help information For example 271 Unipro UGENE Manual Version 1 20 0 ugene help Shows general UGENE CLI help ugene h ugene help lt option_name gt Shows help for the lt option_name gt option ugene h lt option_name gt ugene help lt task_name gt Shows help for the lt task_name gt task ugene h lt task_name gt task lt task_name gt lt task_parameter gt value Specifies the task to run A user defined UGENE workflow schema can be used as a task name For example ugene task align in COI aln out result aln ugene task C myschema uwl in COI aln out res aln log no task progress A task progress is shown by default when a task is running This option specifies not to show the progress log level lt category1 gt lt level1 gt Sets the log level per category If a category is not specified the log level is applied to all categories The following categories are available e Algorithms e Console e Core Services e Input Output e Performance e Remote Service e Scripts e Tasks The following log levels are available TRACE DETAILS INFO ERROR or NONE By default loglevel ERROR For example ugene log level NONE ugene log level Tasks DETAILS Console DETAILS log format lt format_string gt Specifies the format of a log line Use the following notat
231. ment Options Region Custom region 185965 186141 E Indude Left Overhang E Indude Right Overhang Direct Reverse complement Direct Reverse complement lf a primer has been selected you can choose to create the PCR product from this primer Otherwise you can either choose to create the PCR from the whole sequence or choose the Custom item and input the custom region J To add a 5 overhang to the direct strand check the nclude Left Overhang check box and input the required nucleotides To add a 5 overhang to the reverse strand in addition to the described steps select the Reverse complement item in the same group box Similarly to add a 3 overhang check the Include Right Overhang check box input the required overhang and select either the direct or the reverse complement strand On the Output tab of the dialog you can optionally modify the annotations output settings Finally press the OK button to create the PCR product The PCR product will be saved as an annotation In Silico PCR In Silico PCR Overview In silico PCR is used to calculate theoretical polymerase chain reaction PCR results using a given set of primers probes to amplify DNA se quences UGENE provides the In silico PCR feature only for nucleic sequences To use it in UGENE open a DNA sequence and go to the n silico PCR tab of the Options Panel 197 Unipro UGENE Manual Version 1 20 0 Y Forward primer ACGTT ACGTACGTACTACGTACGTGC Tm
232. misc_feature Basically you need to specify the file to read annotations table from required File to read D projects dev ugene trunk test _common_data scenarios annotations_import anns Lcsv And the format of and the path to the file to write the annotations table into required Results Result fle 0 orojects dev ugene trunk test _common_data scenarios annotations_import result gb se fee Check Add result file to project to link the annotations to the currently opened sequence Add result file to project To use a separator to split the table check the Column separator item and specify the separator symbols Also you can press Guess to try to detect the separator from the input file File parsing Colum separator ee Alternatively you can press Edit and edit the script which will specify the separator for each parsed line It is possible to use line number in the script 76 Unipro UGENE Manual Version 1 20 0 Script U Script Editor xam First lines a 2 Script text The script parses input line i and returns an array of parsed elements as the result var line input line var lineNum parsed line number var firstColumn flineNum var otherColumns line split result firstColumn concat otherColumns Using the arrows you exclude the necessary number of lines at the beginning of the document from parsing You can also skip all lines that start with the specified text
233. mple of usage annotations can be exported to this format the Weight Matrix matrices list can also be saved to this format For example it is used to store reports These formats are used throughout the program to save screenshots etc It is possible to view and modify plain text files in UGENE 294 Unipro UGENE Manual Version 1 20 0 Tutorials e Using BioMart with UGENE Environment requirements Installing UGENE extension on Mozilla Firefox Opening data found using BioMart in UGENE Opening BioMart data in UGENE by ID Opening selected data in UGENE Using BioMart with UGENE The BioMart system enables scientists to perform advanced querying of a wide range of biological data sources through a single web interface regardless of the data sources geographical locations This tutorial describes how data found through the BioMart web interface can be easily opened for further analysis in UGENE by a couple of mouse clicks Environment requirements Installing UGENE extension on Mozilla Firefox Opening data found using BioMart in UGENE Opening BioMart data in UGENE by ID Opening selected data in UGENE Environment requirements Currently UGENE extension is available for Mozilla Firefox web browser only Please make sure to launch UGENE before using the extension Follow the instructions below to install the extension Installing UGENE extension on Mozilla Firefox To install UGENE extension on Mozilla Firefox open Add ons Ma
234. mplement reads Use best mode during the alignment Omit reads with qualities lower than The following parameters are available Reference sequence DNA sequence to align short reads to This parameter is required Result file name file in UGENE database format or SAM format if the box SAM output check to write the result of the alignment into This parameter is required Prebuilt index check this box to use an index file instead of a reference sequence Also you can build it manually SAM output checking this box allows one to save output files in the SAM format The default format of output files is the UGENE database format ugenedb Short reads each added short read is a small DNA sequence file At least one read should be added i The Aligning Short Reads with UGENE Genome Aligner has no limitation on short reads length Common parameters Mismatches allowed check this box to allow mismatches between the reference sequence and a short read Select one of the following 239 Unipro UGENE Manual Version 1 20 0 e Mismatches number to set the number of mismatched nucleotides allowed This parameter can take values 1 2 and 3 e Percentage of mismatches to set the number of mismatches in percents Note that in this case the absolute number of mismatches can vary for different reads This parameter can take values 1 10 Align options e Use GPU optimization use an openCL enabled GP
235. n in the left part of the Sequence overview is pressed density of annotations in the sequence is shown For example in the picture below there are annotations in the parts of the sequence that are marked with dark grey color 24k 22k zk See also e Sequence Zoom View e Sequence Details View Sequence Zoom View The Sequence zoom view is designed to provide flexible tools for navigation in large annotated sequence regions 49 Unipro UGENE Manual Version 1 20 0 The most Sequence zoom view space is used to visualize annotations for the sequence The annotations are organized in rows by their names If two annotations with the same name overlap an extra row is created For every row the name and the total number of annotations in the row are shown with a light grey text at the left part of the area NC_014267 dna OA il as rl Z 10k 20k 30k 40k 50k 60k 70k BOk 4 901 100k 110k 120k 130k140 426 a Louw SSC small 3ingle copy E Name and number of annotations in the row a a tel Eme Zooming Annotations repeat region eh Ruler cya E TNE ae 56 167 60k 62k 64k 66k 68k 7Ok 72k 74k 76k 78k 80k 82k 84k 86k 88k 91274 Fi TT k E Below the annotation rows there is a ruler to show coordinates in the sequence Sequence Details View The Sequence details view is a supplementary component of the Sequence overview It is used to show sequence content without zooming Every time you d
236. n 1 Hepatocyte nuclear factor 1 Hepatocyte nuclear factor 3 Hepatocyte nuclear factor 4 Interferon regulatory factors Interferon stimulation response element MyoD belongs to a family of proteins known as myogenic regulatory factors MRFs Myogenin Neurofibromin 1 Transcription factor NF E2 45 kDa subunit is a protein that in humans is encoded by the NFE2 gene Pre existing component of the NFAT Nuclear factor of activated T cells transcription complex Nuclear factor kappa light chain enhancer of activated B cells The p50 NFKB1 p65 RELA heterodimer is the most abundant form of NF kB The c Rel protein is a member of the NF kB family of transcription factors and contains a Rel homology domain Nuclear transcription factor Y Nuclear factor erythroid derived 2 like 2 Octamer transcription factor 1 Octamer transcription factors Protein 53 Paramedian pontine reticular formation Is a protein that in humans is encoded by the SPI1 gene CAMP response element binding CAMP response element binding Serum response element Serum response factor Signal Transducer and Activator of Transcription 1 Signal Transducer and Activator of Transcription 207 Tir USF yy1 Prokaryotic Name AgaR AgaC ArcA ArgR CpxR Crp CysB CytR DeoR DnaA FadR fis FInDC Fnr Frur FUR GALR GALS GLPR GNTP HNS ICLR IHF ISCR1 ISCR3 LEXA Unipro UGENE Manual Version 1 20 0 Thyroid transcription
237. n 65 Other parameters Maximum number of word matches t an upper limit of word matches between a read and other reads Increasing the value would result in more accuracy however this could slow down the program The specified value should be more than 0 Band expansion size a a number of bases to expand a band of diagonals for an overlapping alignment between two sequence reads The specified value should be more than 10 Max gap length in any overlap f reject overlaps with a gap longer than the specified value A small value may cause the program to remove true overlaps and to produce incorrect results This option may be used by the user to split reads from alternative splicing forms into separate contigs The specified value should be more than 1 Assembly reverse reads r consider reads in reverse orientation for assembly The default value is checked SPAdes SPAdes St Petersburg genome assembler Click this link to open SPAdes homepage SPAdes is embedded as an external tool into UGENE Open Tools DNA assembly Settings Window Help m3 DNA assembly t Convert UGENE Assembly database to SAM format mre Align to reference gt Assembly genomes TEN HMMER tools Contig assembly with CAP3 ae Multiple alignment ee ee Select the Assemble genomes item to use the SPAdes The Assemble Genomes dialog will appear 243 Unipro UGENE Manual Version 1 20 0 U Ass
238. n see the description of the annotation saving parameters here Search timeout the remote task terminated if the timeout is reached There is a little difference in default values of parameters between NCBI Nucleotide BLAST web interface and UGENE e The web interface uses the megablast option by default the search is fast but only highly similar sequences are found 176 Unipro UGENE Manual Version 1 20 0 e UGENE ignores the option by default the search may take more time but all somewhat similar sequences are found Check the Megablast option if you want exactly the same results to be found in UGENE as you had in the NCBI web interface Also there is Advanced options tab A Search Through a Remote Database General options Advanced options Word size Match scores Entrez query Filters Low complexity filter Human repeats filter Filter results Filter by E accession E def filter by definition of annotations E id Gap costs Masks E Mask for lookup table only E Mask lower case letters Select result by Evalue Score The view of the Advanced options tab depends on the selected search For the blastn search it looks like on the picture above Word size the size of the subsequence parameter for the initiated search Gap costs costs to create and extend a gap in an alignment Increasing the Gap costs will result in alignments which decr
239. n the field nearby Note that this value also depends on the pattern length and is disabled when the pattern hasn t been specified e Substitute a pattern may contain characters different from the characters in the searched region When this algorithm has been selected you can also specify the match percentage and additionally it is possible to take into account ambiguous bases e Regular expression a regular expression may be specified instead of a pattern For example character matches any character 2 matches zero or more of any characters There is also the Limit result length option that specifies the maximum length of a result e Exact find a place where one or several patterns are found within a larger pattern Search in 7 Search in Strand Both Search in Sequence Region se In this group you can specify where to search for a pattern in what region and in which strand for nucleotide sequences Also for nucleotide sequences it is possible to search for a pattern on the sequence translations Strand for nucleotide sequences only Specifies on which strand to search for a pattern Direct Reverse complementary or Both strands Search in for nucleotide sequences you can select the Translation value for this option In this case the input pattern will be searched in the amino acid translations Region specifies the sequence range where to search for a pattern You can search in the whole sequence
240. nager and select Install Add on From File item in the settings menu 295 Unipro UGENE Manual Version 1 20 0 Firefox oe E le x j Add ons Manager nage Check for Updates View Recent Updates A In the browse dialog select ugene xpi file that you can find in the Firefox directory of the UGENE Web Browsers Extensions Package that there is on the Download page Opening data found using BioMart in UGENE For now there are two options to open data found using BioMart in UGENE e Open data by ID for example by an Ensembl ID e Open selected data Opening BioMart data in UGENE by ID Let s open web site 296 Unipro UGENE Manual Version 1 20 0 lt C D www biomart org 0o aN a Community Publications News Bio Portal 45 databases in 4 continents and growing Version 0 8 Proceed to Portal Proceed to Portal Proceed to Portal gt Version 0 8 Click for example on the Proceed to Bio Portal link The following page will appear J 8h BioMart x J 2 BioMart Portal x gt D central biomart org BioMart Central Portal Home IDENTIFIER SEARCH Databases 41 Examples KRAB ENS gas ee ae F CANADA 1 FRANCE UNITED KINGDOM Es 4 21 ITALY SPAIN 1 1 CHINA 1 Gene retrieval Variant retrieval Sequence retrieval ID converter UNITED STATES m Cancer genes Ensembl Ensembl Bacteria Ensembl Fungi Ensembl Meta
241. navailable e g if the reads are from a FASTA file the Phred quality defaults to 40 The v alignment mode In v mode alignments may have no more than V mismatches where V may be a number from 0 through 3 Quality values are ignored The v mode is mutually exclusive with the n mode The following parameters are available Maq error magerr maximum permitted total of quality values at all mismatched read positions throughout the entire alignment not just in the seed The default is 70 By default Bowtie rounds quality values to the nearest 10 and saturates at 30 Note that the rounding can be disabled with No Mag rounding Seed Length seedlen the number of bases on the high quality end of the read to which the n applies The lowest permitted setting is 5 and the default is 28 Maximum of backtracks maxbts the maximum number of backtracks default 125 without Best 800 with Best A backtrack is the introduction of a speculative substitution into the alignment Descriptors memory usage chunkmbs the number of megabytes of memory a given thread is given to store path descriptors in the Best flag Default 64 This parameter is available if the Best flag is checked Seed seed pseudo random number generator The following flags are available Colorspace color the input is read in colorspace colors are encoded as characters A C G T A blue C green G orange T red No M
242. nce between the whole sequence and a sliding window Let f XY frequency of the dinucleotide XY frequency of the nucleotide X Hh gt lt PRN FR 7 Fx Fy p_seq XY p XY for the whole sequence p_win XY p XY for a window The Karlin Signature Difference for a window is calculated by the following formula sum p_seq XY p_win XY 16 e Informational Entropy is calculated from a table of overlapping DNA triplet frequencies The use of overlapping triplets smooths the frame effect Informational Entropy is calculated by the following formula triplet frequency logl10 triplet frequency 10g10 2 104 Unipro UGENE Manual Version 1 20 0 Graph Settings To change settings of a graph select the Graph gt Graph settings item in the graph context menu The Graph Settings dialog appears 2 Graph Settings Window Steps per window Default color M F Cutoff for minimum and maximum values Minimum 0 00 Maximum 0 00 ok Cancel _ The following parameters are available Window the number of bases in a window Steps per window the number of steps in window The Step is calculated as Window Steps per window Default color the default color of line of graph or lines of graphs for GC Frame Plot Checking of the Cutoff for minimum and maximum values checkbox enables the following settings Minimum the minimum value for cutoff Maximum the maximum value for cutoff
243. nce from Alignment Exporting Alignment as Image Undo Redo Framework The editor tracks all modifications of the aligned sequences When a modification happens the current state of the multiple sequence alignments object is being recorded You can apply any previous state and redo the modifications using the corresponding buttons on the toolbar 125 Unipro UGENE Manual Version 1 20 0 WSUS intera 1 Selecting Subalignment While in the Sequence area if you hold the left mouse button and move the cursor you will activate the selection mode By moving the cursor you can adjust the size of the selection Also you can use the Shift modifier for selecting Select a first row hold Shift and select a las row All the rows between the first and the last row will be selected Releasing the mouse button will result in exiting the selection mode The selection mode is available in the Sequence list and the Consensus area too The difference between these areas and the Sequence area is that here you can add to selection the whole rows or columns respectively To cancel the selection press the Esc key Moving Subalignment To move subalignment there are different ways 1 Select a subalignment and drag and drop it The subalignment will be moved 2 With a help of the Spase the subalignment will be moved to the right by size of the selection With a help of the Backspase the subalignment will be returned to the first state 3 W
244. ne Symbols can be changed by clicking on interesting value modifications are shown in bold Also you can show hide different signals of chromatogram and quality bars with a help of the Show hide trace and Show quality bars toolbar buttons correspondingly 99 Unipro UGENE Manual Version 1 20 0 ol Show quality bars ATOEPTAA CC AG TTC Showlhide trace e Exporting Chromatogram Data e Viewing Two Chromatograms Simultaneously Exporting Chromatogram Data Open for example the UGENE data samples SCF 90 JRI 07 srf file In the Project View context menu there is Export chromatogram to SCF item Project x OY 30 JRI 07 sequence Objects 90 JR1 O jee 9 s at Open view d Chromatogr am 9 c gi Add ko sjem HL Unload selected documents Lock document For editing Add d Export sequences Remove d Export sequences as alignment UIMencs Export chromatogram to SCF 1 iW 20 30 40 50 BO r0 Name After clicking on the item the Export chromatogram file dialog will appear m Export Chromatogram File Export to file C work ugene data samples SCF 90 JRI O7_copy scf Reversed Complemented Add document to the project Check the Reversed and Complemented options if you want to create a reverse and complement chromatogram Press the Export button The exported file will be opened in the Sequence View 100 Unipro UGENE Manual Version 1 20 0 Viewing Two Ch
245. ng a protein query e blastx searches a protein database using a translated nucleotide query e tblastn compares a protein query against a translated nucleotide database the all six reading frames e tblastx translates the query nucleotide sequence in all six possible frames and compares it against the six frame translations of a nucleotide sequence database e makeblastdb formats protein or nucleotide source databases before these databases can be searched by other BLAST tools BLAST home page hittp blast ncbi nim nih gov Blast cgi CMD Web amp PAGE_TYPE BlastHome To make BLAST or BLAST tools available from UGENE 1 Install the required verion of BLAST or BLAST on your system 2 Set the paths to the executables you are going to use on the External tools tab of UGENE Application Settings dialog After you ve finished this configuration you can access the tools from the Tools BLAST submenu of the main menu e Creating Database e Making Request to Database e Fetching Sequences from Local BLAST Database Creating Database To format a BLAST database do the following e If you re using BLAST open Tools BLAST FormatDB e If you re using BLAST open Tools BLAST BLAST make DB The Format database dialog appears 180 Unipro UGENE Manual Version 1 20 0 m Format Database Input data Select input file s for formatting database Or select directory with input files File filter
246. ng the button Set recognition bound on the toolbar 261 Unipro UGENE Manual Version 1 20 0 T UGENE Expert Discovery File Actions Settings Tools Window Help Seb RBbB EA 8 e The following dialog will appear T Setup Recognition Bound Recognition Bound Optimize Recognition Bound Information Probability of negative sequence recognition Probability of positive sequence rejection Recognition Graph i P F o b a b l i t Y 10 12 14 16 18 20 2 24 25 268 30 32 HY JS J5 40 42 44 4 4850 Score In the dialog errors of the first and the second type are shown for choosing the value Also for convenience an HTML recognition report can be generated The report includes statistical parameters and a recognition result for each sequence 262 Unipro UGENE Manual Version 1 20 0 Shared Database The rational storage of biological data is an ever present issue It is not only about large data sizes but also about the requirement of simultaneous access to them by several scientists For instance a few researchers from a lab may need to work on the same data like a set of primers or data produced by sequencing That information has to be updated and synchronized between different users and kept ina common storage That is what UGENE Shared Database is intended for a LEY To start sharing data via UGENE you need to deploy a public database server MySQL servers are currently sup
247. ngs are available Branches width Height Hide pen settings cr Line weight 1 ml Here you can select the color and the line width of the tree branches Note that when a clade has been selected the branch settings are applied to the clade only Zooming Tree To change the size of a tree use the Zoom In and Zoom Out toolbar button You can use the Restore Zooming toolbar button to set the default size Or use the corresponding items in the Actions main menu See also Zooming Clade 160 Working with Clade This paragraph describes how to select a clade and modify it s appearance Selecting Clade Collapsing Expanding Branches Swapping Siblings Zooming Clade Adjusting Clade Settings Changing Root Selecting Clade To select a clade click on it s root node You can see that the corresponding branches are highlighted Unipro UGENE Manual Version 1 20 0 To select several clades at the same time hold the Shift key and click on the root nodes of the clades Collapsing Expanding Branches You can hide branches of a clade by selecting the Collapse item in the context menu of the clade s root node or use the Collapse button on the tree toolbar 161 Unipro UGENE Manual Version 1 20 0 COl16 nwk X R O s OC oun Be vs The branches have been collapsed Zychia_baranovi Deracantha_deracantoides EF540 0 067 Tettigonia viridissima 4 Fodisma_sapporensis Zoom In
248. nize and lock visual ranges of different sequences shown in the Seq uence View This feature is available when there are two or more sequences opened in the same Sequence View If we click the Lock scales button the second sequence scale will be adjusted to be the same as the focused sequence scale and is locked Now if we move a scrollbar or use zoom buttons for any of the sequence visual ranges for the rest sequences will also be adjusted Lock scales button Synchronize kk Ss with out lock F Y L p p Scales button i i To unlock the scales click the same button again You may use the Adjust scales button to synchronize scales without locking them Note that if you have a selected sequence region or a selected annotation the scales will be synchronized by the start position of the region or the annotation If there are no active selection the regions are synchronized by the first visible sequence position on the screen Multiple Sequence Opening To open several sequences use the File gt Open menu item or Open toolbar button and using Ctrl select the several sequences and click the Open button The following dialog will appear 65 Unipro UGENE Manual Version 1 20 0 m Multiple Sequence Reading Mode Separate sequence mode C Merge sequence mode Number of unknown symbols N for nucdeic acid or X for amino acid between sequences 10 Join sequences into alignment New document name Save document
249. nment For gapped alignment X dropoff value in bits for gapped alignment For ungapped alignment X dropoff value in bits for ungapped alignment For final gapped alignment X dropoff value in bits for final gapped alignment Multiple hits window size multiple hits window size Perform gapped alignment performs gapped alignment 185 Unipro UGENE Manual Version 1 20 0 Fetching Sequences from Local BLAST Database To fetch sequences from local BLAST database use the Fetch sequences from local BLAST database gt Fetch sequences by id from blast result context menu item of the blast result The following dialog will appear m Fetch Sequence from BLAST Database Entery query ID Select database Type of file s nucdeotide Output path Add to project Fetch Here you need select a query ID database type of file s and output path After that click on the Fetch button To fetch sequences for several annotations at the same time select the blast results with Ctrl key and call the Fetch sequences from local BLAST database gt Fetch sequences by id from blast result context menu item Repeat Finder The Repeat Finder plugin provides a tool to search for direct and invert repeats in a DNA sequence Also it allows to search for tandem repeats e Repeats Finding e Tandem Repeats Finding e Tandem Repeats Search Result Repeats Finding Usage example Open a DNA sequence in the Sequen
250. nt into another file click the Save alignment as button uU UGENE BLO60C3 m Contig1 File Actions Settings Tools Window Help a e ii mA a t W Fa i k Aligning Sequences The Alignment Editor integrates several popular multiple sequence alignment algorithms Below is the list of available algorithms and links to the documentation 127 Unipro UGENE Manual Version 1 20 0 e Port of the popular WUSCLE3 algorithm e KAlign plugin effective work with huge alignments e ClustalW and MAFFT these algorithms appeared in the version 1 7 2 of UGENE with the External Tools plugin e T Coffee this alignment algorithm is available since version 1 8 1 of UGENE with the External Tools plugin To align sequences choose a preferred alignment method in the Actions main menu in the context menu or by Align main toolbar button Also you may find useful the following video tutorials devoted to the multiple sequence alignment e Making a multiple sequence alignment from FASTA file e Working with large alignments in UGENE e Performing profile to profile and profile to sequence MUSCLE alignments e Running remote MUSCLE task Aligning Sequence to this Alignment To align a Sequence to an opened alignment click the Align sequence to this alignment toolbar button il U uy ea ll JE Choose a file with the sequence from the files system and click Open elf Also you can add an already opened sequence or
251. ntext menu The Smith Waterman Search dialog appears a Smith Waterman Search Smith Waterman parameters Input and output Enter pattern here Search in Strand Sequence F Both Translation Direct Complement Smith Waterman algorithm parameters Algorithm version Scoring matrix Gap scores Results filtering strategy weon a0 E _ Repertresuts RrnaecinSL gt Advanced Gap extension 1 gt Minimal score First of all you need to specify the pattern to search for The rest parameters are optional Search in select either to search in the sequence or in its translation Strand select the strand to search in direct complementary or both strands Region specifies the region of the sequence that will be used to search for the pattern By default if a subsequence has been selected when the dialog has been opened then the selected subsequence is searched for the pattern Otherwise the whole sequence is used You can also input a custom range 210 Unipro UGENE Manual Version 1 20 0 Algorithm version version of the algorithm implementation Non classic versions produce the same results as classic but much faster To use these optimizations our system must support these capabilities e Classic 2 SSE2 e CUDA e OPENCL Scoring matrix can be chosen from a bunch of matrices supplied with UGENE To view a matrix selected click the View button Gap open penalty for opening a gap Gap extension
252. ntext menu item in the Sequence area or use hot key combination Note that if you activate context menu in the Sequence list area you will lose your current selection Ctrl s c 0 bo position Add Copy Copy selection mae Ga Colors d Copy consensus Edit d Copy consensus with gaps 32i Align To copy consensus sequence use the Copy Copy consensus item Renaming Sequences To rename a sequence double click on the name of this sequence and enter a new sequence name in the dialog Sorting Sequences To sort sequences by name in the alphabetical order choose the View Sort sequences by name item from the Actions main menu or the context menu Shifting Sequences To change an order of sequences in a multiple sequence alignment do the following e select sequence or sequences in the sequences names list by click or by click and drag correspondingly e click and drag on selected region to shift it Collapsing Rows It is able to coolaps the sequential rows To collapse rows click on the Switch on off collapsing main toolbar button D G The triangle will appear near collapsed sequences Click on the triangle to show the whole tree of the collapsed rows 130 Unipro UGENE Manual Version 1 20 0 Consensus Bicolorana_bicolor_EF540830 Conocephalus_discolor Conocephalus_percaudata Conocephalus_sp Deracantha_deracantoides_EF5S40 Gampsocleis_sedakovii_
253. o remove specifies the region of the sequence that will be removed in the form This parameter is mandatory Annotated regions resolving mode specifies what to do with annotations that overlap with the region that is removed You can select either Crop corresponding annotation i e make it smaller or Remove corresponding annotation Recalculate values of qualifiers recalculates regions in qualifiers when sequence is modified Save to new file similar to the same parameter in the nsert subsequence dialog described above Also it is possible to invert sequence When you select the Reverse complement sequence Complement sequence or Reverse sequence ite ms in the context menu or in the Actions menu the sequence will be inverted correspondingly Exporting Selected Sequence Region Open a sequence object in the Sequence View and select a region by pressing and moving the left mouse button over the sequence Use the Export Export selected sequence region context menu item to save selection into a file of a sequence format 63 Unipro UGENE Manual Version 1 20 0 UGENE murine is NC_001363 File Actions Settings Tools Window Help GOR 4 w a i ga a 0P NC 001363 dna Goto position Ctrl G Select sequence region Ctri A New annotation Ctrl N Rename item Copy Paste Select Add Analyze Align Cloning Export Edit Export sequence of selected annotations Remove Export annotation
254. o the following right click on the installer exe le and select Run as administrator item Alternatively to use UGENE without installing e Download UGENE zip package ae Windows Packages for Windows XP Windows Vista Windows and higher Windows versions Installers e Download 32 bit Standard or Full installer package Download 64 bit Standard or Full installer package ip bundels Download 32 bit portable Standard or Full zip bundle Download 64 bit portable Standard or Full zip bundle Download 64 bit NGS portable zip bundle caution zip bundle size is about 4Gb e Unpack it e Launch the ugeneui exe le Installation on Mac OS X e Download the Mac OS X Disk image le using the appropriate link on the download page Unipro UGENE Manual Version 1 20 0 m amp MacOSX Packages for Mac OS X 10 5 and higher Download 32 bit Standard or Full package for Mac OS X 10 5 and higher Download 64 bit Standard or Full package for Mac OS X 10 6 and higher Download 64 bit NGS package caution package size is about 4Gb for Mac OS X 10 6 and higher Also find below UGENE packages for old Mac OS X versions Note that they updated from time to time not in each release PowerPC the latest available version is 1 10 3 download Mac OS X 10 4 Tiger Intel the latest available version is 1 10 3 download e Launch the dmg le and accept the GNU license agreement The following wind
255. o you can translate sequence s to amino alphabet Also it is possible to specify whether to merge the exported sequences into a single sequence or store them as separate sequences If you merge the sequences you re allowed to select the gap symbols between sequences This is the length of the insertion region between sequences that contain N symbols for nucleic or X for protein sequences Export sequence with annotations 28 Unipro UGENE Manual Version 1 20 0 To export sequence with annotations choose Genbank or GFF format The Export with annotations checkbox will ba available Check the checkbox and sequence will be exported with annotations Exporting Sequences as Alignment Suppose we want to interpret FASTA file as multiple alignment To do this select a single or several sequence objects in the Project View wi ndow click right mouse button to open the context menu and select the Export Export sequences as alignment item oa UGENE fasta_example File Actions Settings Tools Window Help Oo SB 4 SG a Ga Ea Ea Project Name filter of Objects om 4 if fasta_example 4 s Phaneroptera_falcata Is _altaica_EF540820 s Bicolorana_bicolor_EF540830 E al s Roeseliana_roese rm oe I F A E 4 s Montana_montz Open view TCTAATTCGACCCGAATTAG k bia da Add to view 8 10 12 14 16 18 20 22 24 27 E s Gampsocleis_sec Unload selected objects AGATTAAGCTCGGCTTAATC s Deracantha_deriz A s Zychia_ba
256. ode is much slower than the default You can also configure the following advanced parameters Enable long gaps checking this box allows one to set the Max gap extentions parameter Max gap extensions e maximum number of gap extensions Indel offset i disallow insertions and deletions within the specified number of base pairs towards the ends Max long deletion extensions d disallow a long deletions within the specified number of base pairs towards the 3 end Max queue entries m maximum queue entries Barcode length B length of barcode starting from the 5 end When the specified length is positive the barcode of each read will be trimmed before mapping and will be written at the BC SAM tag For paired end reads the barcode from both ends are concatenated Threads t number of threads Max seed differences k maximum edit distance in the seed Mismatch penalty M BWA will not search for suboptimal hits with a score lower than the specified value Gap open penalty O gap open penalty Gap extension penalty E gap extension penalty Quality threshold q parameter for read trimming 231 Unipro UGENE Manual Version 1 20 0 Select the required parameters and press the Start button Building Index for BWA To build BWA index select the Tools DNA Assembly Build Index item in the main menu The Build Index dialog appears Set the Align short reads method parameter
257. ofile can be used as a set of sequences The result of such sequences to profile alignment is presented on the picture below Consensus ATGGcaCatcCCctcaCAac tAGGttTtrtC AAG Ac GCagqgccTcacCca 1 k m m m m m m m j _ j j j j _ _ j _ jl _ jl T gt Ts T gt E y I gt f gt T z The original alignment is not modified only columns with gap character can be inserted The second profile was considered as a set of sequences and therefore is modified Note that if a file with another alignment is used as a source of unaligned sequences the gap characters are removed and each input sequence is processed independently This method is quite fast for example an alignment of 3000 sequences 1000 bases each to the existing profile takes about 5 minutes on the usual Core2Duo computer ClustalW Clustal is a widely used multiple sequence alignment program It is used for both nucleotide and protein sequences ClustalW is a command line version of the program 221 Unipro UGENE Manual Version 1 20 0 Clustal home page http www clustal org If you are using Windows OS there are no additional configuration steps required as ClustalW executable file is included to the UGENE distribution package Otherwise e Install the Clustal program on your system e Set the path to the ClustalW executable on the External tools tab of UGENE Application Settings dialog Now you are able to use Clustal from UGENE
258. ollowing dialog appears m Shared Databases Connections UGENE public database Connect Disconnect To add new connection click on the Add button The following dialog appears 264 Unipro UGENE Manual Version 1 20 0 Database location Authentication data Host Port Database Here you need to specify Host IP address of the server Port number of the port used by the MySQL server and Database name of the database You may also fill Login and Password fields Otherwise you are asked to input them every time you are establishing this connection until you check the Remember me box Click on the OK button then the connection is created and the appropriate item appears in the previous Shared Database Connections dialog If you want to use already existing connection choose the appropriate item in the Shared Database Connections dialog and press the Conne ct button This can also be done by double clicking the item If the specified database is empty UGENE has to initialize it This routine is done only once In this case you get an appropriate message box asking whether to initialize the database or not If you choose Yes the database is populated with UGENE data structures if No it remains empty and UGENE does not connect to it If you want to delete some connection select it in the Shared Database Connections dialog and click on the Delete button You may also edit connection parameters using the Edit button An estab
259. ological sequence analysis usin oa HMM 3 MIM profile tools Plugin is base a Kalign On 4 port of Kalign package For mul a MUSCLE On 4 port of MUSCLE package For a ORF Marker Oni Finds open reading Frames ORF oa Optimized Smith Wwaterm On Various implementations of Smit oa Phylip plugin Oni PHYLIP the PHY Logeny Inferenc oa Primer 3 On Integrated tool for PCR primers a PsiPred On PsiPred protein secondary struc The window shows the list of available plugins To add or remove plugins use the Add plugin and the Remove plugin items available in the Plugin Viewer context menu On 4d plugin Remove plugin Direct SOCKEE Transport When you select the Remove plugin item for a plugin the plugin s status is changed to the to remove after restart value The Remove plugin s no more available in the context menu of the plugin Instead the Enable plugin item appears in the context menu 35 Unipro UGENE Manual Version 1 20 0 If you select this item the plugin will be enabled again i e it will not be removed after restart Otherwise the plugin will not be available after UGENE restart Searching NCBI Genbank UGENE allows searching data in NCBI GenBank remote database To do this open the following dialog by File gt Search NCBI Genbank main menu T NCBI Sequence Search Term All fields os Database nucleotide Search query History
260. olumn highlighting are available from the Reads shadowing item in the context menu of the Reads Area e Disabled highlights all columns of nucleotides e Free highlights all reads that intersect a given column In this mode you can lock a position Click the Lock here item in the context menu to do it To return to a locked position select the Jump to locked base item in the context menu e Centered highlights all reads that intersect the column in the center of the screen 148 Unipro UGENE Manual Version 1 20 0 Lo be i E EET ET TF O A A a a a a Associating Reference Sequence To associate a reference sequence with the assembly open the sequence the sequence must be loaded and drag it to the Assembly Reference Area Objects 23 738 to 27 954 4 216 bp as pkF70 as pkfl40 as pKFo4 4 E pKF3 all fa s pkF70 s pkf140 2 s pKFo4 27 399 C29 The sequence appears in the Reference Area 23 738 to 27 954 4 216 bp 2r oo To remove the association select the Unassociate item in the Reference Area context menu Associating Variations To associate variations with the assembly open the sequence the sequence must be loaded and drag it to the Assembly Reference Area 149 Unipro UGENE Manual Version 1 20 0 0 to 16 571 16 571 bp 4 if chrM fa ka s chrM a H chrM sam ugenedb 9 as chrM H exportedToSNEsnp v chri var
261. om sequence name To do it check the corresponding checkboxes Use the Conversion options to choose a strand for saving sequence s Also you can translate sequence s to amino alphabet Also it is possible to specify whether to merge the exported sequences into a single sequence or store them as separate sequences If you merge the sequences you re allowed to select the gap symbols between sequences This is the length of the insertion region between sequences that contain N symbols for nucleic or X for protein sequences Export sequence with annotations To export sequence with annotations choose Genbank or GFF format The Export with annotations checkbox will ba available Check the checkbox and sequence will be exported with annotations Using Bookmarks One of the most important features supported by most Object Views is an ability to save and restore visual view state Saving and restoring visual state of an Object View enables rapid switching between different data regions and is similar to bookmarks used in Web browsers Initially an Object View is created as transient It means that its state is not saved To save current state of a view select an item with the view name in the Bookmarks part of the Project View windows and select the Add bookmark item in the context menu 32 Bookmarks oo WF Lay MI_uvolee reacures g9 s NT_OV8122 sequence Unipro UGENE Manual Version 1 20 0 dL Ll E aLi a hs_chr NT_011
262. on The Source URL field in the dialog specifies the file to import The Destination URL field specifies the output database file Browsing and Zooming Assembly Opening Assembler Browser Window Assembly Browser Window Assembly Browser Window Components Reads Area Description Assembly Overview Description Ruler and Coverage Graph Description Go to Position in Assembly Using Bookmarks for Navigation in Assembly Data Opening Assembler Browser Window An imported assembly added to the project is shown in the Project View as follows Project x Name filter 4 E Klebsislla sort bam ugenedb as pkF70 as pkfl40 Each as object corresponds to an imported contig When you double click on an as object a new Assembly Browser window with the assembly data is opened A window for the first assembly object in the list is opened automatically after the import Assembly Browser Window 143 Unipro UGENE Manual Version 1 20 0 The opened window contains the list of well covered regions of the assembly O to 16 571 16 571 bp 1 to 16 571 16 571 bp gt 10k 15 385 C 259 a zoom in to see the reads or choose one of the well covered regions 1i 6950 330 2 7950 321 3 16250 z320 4 8650 316 3 16 350 312 6 15450 308 7 12950 307 11750 301 9g 127050 300 i0 13 250 296 TIP Page up Page down Move one page up down in the Reads Area Note that for large assemblies it may take some
263. on utilizing modern multi core processors The current version of UGENE provides user interface for three HMM3 tools HMM3 build HMM3 search and Phmmer search In the original program the corresponding commands are hmmbuild hmmsearch and phmmer To access these tools select the Tools HMMER3 tools submenu of the program main menu Window Help i E Create index file es DNA Assembly Foe Weight matrix Ba HMMERZ tools _ 89 SITECON i LCE Build profile workflow Designer Search HMM signals Fhmmer search TT Fs m sir T T T sma Tr T T We highly recommend reading the original HMMER3 documentation to learn how to use utilities provided by the plugin e Building HMM Model HMM3 Build e Searching Sequence Using HMM Profile HMM3 Search e Searching Sequence Against Sequence Database Phmmer Search Building HMM Model HMM3 Build The HMM3 build tool is used to build a new HMM profile from a multiple alignment You can use any alignment file formats supported by UGENE The output HMM profile format is compatible with the HMMER3 package but it is not compatible with the HMMER2 The HMM3 build automatically calibrates the target model T Hmm3 Build Input and output Construction strategies Input alignment file Build to profile The HMM3 configuration dialog provides an easy way to set appropriate search parameters Here you can see effective weighting strategies options 215 Uni
264. ong genomes Score for a match a score of a match Mismatch penalty b mismatch penalty Gap open penalty q gap open penalty Gap extention penalty r Gap extension penalty The penalty for a contiguous gap of size k is q k r Band width w Band width in the banded alignment Number of threads t Number of threads in the multi threading mode Size of chunk of reads s Maximum SA interval size for initiating a seed Higher s increases accuracy at the cost of speed Score threshold divided by much score T minimum score threshold Z best z Z best heuristics Higher z increases accuracy at the cost of speed Number of seeds to start rev alignment N Minimum number of seeds supporting the resultant alignment to skip reverse alignment Mask level c Coefficient for threshold adjustment according to query length Given an I long query the threshold for a hit to be retained is a max T c log I Prefer hard clipping in SAM output H use hard clipping in the SAM output This option may dramatically reduce the redundancy of output when mapping long contig or BAC sequences Select the required parameters and press the Start button Building Index for BWA SW To build BWA SW index select the Tools Align to reference Build Index item in the main menu The Build Index dialog will appears Set the Align short reads method parameter to BWA SW The dialog looks as follows 234 Un
265. onita EF S083 1 Gampseckis sed fychia baram Changing Root To change root of a tree select the root and call the Reroot tree context menu item or use the Reroot tree button on the tree toolbar COl16 nwk X ROl t amp l Bw Yt 0 076 Metrioptera_ japonica EF 540831 m a a T 0 052 Bicolorana bicolor EF540850 0 009 0 023 T Tae Roeseliana_roeseli Zoom In Te Montana_montana aoe Gampsodeis_sedakovii_FF540328 5 Swap Siblings Turhia hsrarrwi 0 078 Fychia_baranovi Deracantha_deracantoides_EF540 0 067 Tettigonia viridissima Podisma_sapporensis Hetrodes pupus EF540832 Mecopoda_ elongata Ishigaki_ J Mecopoda_elongata_ Sumatra_ Mecopoda_sp _ Malaysia_ Isophya_altaica_EF540820 0 007 Fhaneroptera _falcata Exporting Tree Image A tree image can be exported to a raster format png jog omp etc or to a vector format svg Select either the Export Tree Image toolbar button or the Actions Export Tree Image item in the main menu In the submenu appeared select the Screen Capture item to save the tree image to a raster format The standard Save As dialog will appear where you can select the file name and format To export a tree image to a vector format select the As SVG item in the Export Tree Image submenu Printing Tree To print a tree select either the Print Tree toolbar button or the Actions Print Tree item in the main menu 163 Unipro UGENE Manual Version 1 20 0 The standa
266. ons My Document 11 96 S misc_feature 0 3 E misc_feature 713 1730 misc_feature 1728 1737 m misc Feature 743 175 J S00 NC 01343 feabires Imarine nhi Another way to select a sequence around annotations is to hold Shift and Ctrl keys while clicking on the annotations either in the Sequence details view or in the Sequence zoom view Copying Sequence 57 Unipro UGENE Manual Version 1 20 0 The selected sequence region an annotation sequence or their amino translations can be copied to clipboard e By pressing the corresponding buttons in the global toolbar Actions to copy selected sequence regions i e Using the following shortcuts e Ctrl C copies direct sequence strand e Ctrl T copies direct amino translation e Ctrl Shift C copies reverse complement sequence e Ctrl Shift T copies reverse complement amino translation e Using the Copy submenu of the context menu d Fe N a a T a Goto position Ctrl 3 l TE 2 Select sequence region Ctrl gt fy New annotation Ctrl M oF L L WV EK D M E 5 Copy sequence Ctrl C Select Copy complement sequence Ckrl ShiFt 2 ia Add t Sa Copy translation Ctrl T Analyze bP Sy Copy complement translation Ctrl ShiFt T res s Align d 5A Copy annotation sequence 0 Export Za Copy annotation sequence translation Edit sequence e a Remove d re 24 Rulers O Disable misc Featu
267. ons To invert the selection use the nvert annotation selection item in the Annotations editor context menu Editing Annotation If the document is not locked it is possible to edit an annotation or an annotation group using the Rename item context menu from the Annot ation Editor or from the Sequence View or with a help F2 key in the Annotation Editor The result of pressing for an annotation Wy Edit Annotation Annotation name comment Location 1 5833 The result of pressing for an annotation group Highlighting Annotations To configure settings of annotation names go to the Annotation Highlighting tab in the Options Panel By default the tab shows annotations names of the opened Sequence View Annotations Highlighting Lok 2k 25k Sk aw AE Select an annotation name Annotation Color 1 5k 2k Bad i BaeGl C TEE Bael E a CDS E GACCATCCTCTAGACTGACATGGCGCATTCAACGCCATG 3 L A 3826 3950 3865 3670 Seo 3880 3885 38903894 comment m CTGGTAGGAGATCTGACTGTACCGCGTAAGTTGCGGTAC misc feature L T E 7 y CH a M E W ee yi O M E m Mu i i source l R a L2 A M Show all annotation names q j Configure the annotations Mame Tonle l YP Show annotations E CDS CDS EB CDS CDS Show on translation gt CDS CDS Show value of qualifier E CDS CDS label note re comment 0 1 l ve misc feature 0 2 FF mic feature Mise D misc_feature Misc gt re source 0 1 ui 4
268. ons Settings Tools Window Help Editor toolbar n o Ali ae eae ala et oags amp Consensus area YA Pairwise Alignment Consensus w Sequences TaAGttTatTaATtCGagCtGAAtTagG a F e r en ee eee ee ee ee E ee ee a ee ees De ee E Se ee T eee Select and add 123 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Select and add 29 29 29 29 29 29 29 29 29 1 Project Isophya_altaica_EF540820 Bicolorana_bicolor_EF540830 Roeseliana_roeseli Montana_montana Metrioptera_japonica_EF54083 Gampsocleis_sedakovii_EF540 Deracantha_deracantoides_EF5 Zychia_baranovi Tettigonia_viridissima Conocephalus_discolor Conocephalus_sp Conocephalus_percaudata Mecopoda_elongata__Ishigaki_ Mecopoda_elongata__Sumatra_ Mecopoda_sp __ Malaysia_ Podisma_sapporensis Hetrodes _pupus_EF540832 w Output settings In new window Output file 3 PairwiseAlignmentResult aln aas 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sequence area l An Ending offset Coordinates Pa sec RL cl lille Find ee Options panel il ie Mi qtr rey I Mij Th fw i ti inu ier Mm i I a pe i i mm The Alignment Editor components For example let s assume that the coordinate of the first visible base of the row is N but the row contains K gaps before the position N The starting offset value will be N K The same rule is true for the ending offset
269. oolbar press the Set background color button and select a color in the dialog appeared Selecting Detail Level To select the detail level of a 3D Structure representation open the Settings dialog of the 3D Structure Viewer and drag the Detail level slider Enabling Anaglyph View UGENE allows you to view a molecule in the anaglyph mode To enable the anaglyph view open the Settings dialog of the 3D Structure Viewer and check the Anaglyph view check box You can modify the color settings select one of the available Glasses colors or set custom colors swap the colors The offset of the color layers can be adjusted by dragging the Eyes shift slider Eyes shift Glasses colors Cyan Red Left Right a 4 Swap colors See the result the anaglyph view is applied to a molecule below 93 Unipro UGENE Manual Version 1 20 0 Moving Zooming and Spinning 3D Structure A 3D structure can be easily spinned moved and resized e To spin the 3D structure drag the mouse on the 3D structure while holding the left mouse button e To move the 3D structure hold the Ctrl keyboard button and drag the mouse with the left button pressed e To resize the 3D structure either use the mouse wheel or Zoom In and Zoom Out buttons on the toolbar At any time you can restore the default view by pressing the Restore Default View button on the toolbar a You can also overview the whole structure by spinning it automatically Select the Spin item e
270. ory by default String toolpath External tool path using the path specified in UGENE by default String out type Type of BLAST output file using XML m 7 by default String Example ugene gene by gene in human_Tl fa out human_Tl_report Reverse Complement Converting Sequences Task Name revcompl Convert input sequence into its reverse complement or reverse complement counterpart and write result sequence to file Parameters type Type of operation Available are Reverse Complement Complement and Reverse using Reverse Complement by default String in Input file Url datasets accumulate Accumulate all incoming data in one file or create separate files for each input In the latter case an incremental numerical suffix is added to the file name using True by default Boolean format Output file format using fasta by default String split Split each incoming sequence on several parts using 1 by default Number out Output file String Example ugene revcompl in human_Tl fa out human_Tl_result fa format fasta type sreverse Variants Calling Task Name snp Call variants for an input assembly and a reference sequence using SAMtools mpileup and bcftool Parameters bam Input sorted BAM file s Url datasets ref Input reference sequence Url datasets wout Out file with variations String bN A C G T only Boolean 287 bl List of sites String
271. otations chromatograms 3D models statistical data etc Annotation Additional information about a sequence identified by its name and the sequence region Alignment Editor An Object View used to visualize and edit DNA RNA or protein multiple sequence alignments Options Panel An Options Panel it is the panel with different information tabs and tabs with settings for Sequence View and Assembly Browser In the image below you can see a typical UGENE window with a Project View and a single Object View window opened UGENE A File Bo kh 4 Actions Settings Tools Window Help a so Ga En Ea lt a o 7 Information Project view at Length 5833 Options panel ee Fit Characters Occurrence gt By AFI77870 emb Object views gt E human Tifa sequence with annotation f 6 easfastq l E E m p p 17 gt B 1CF7 PDB E i i 4 E murine gb a NC_001363 features s NC_001363 gt E ty3 aln gz A 1427 24 5 1673 1484 125 q 1 Project G T H Dinudeotides 2 4 6 amp 10 12 14 16 18 20 22 M2 TTTACTTTCTGGGGTGOGGCATCCACCE L Annotations Highlighting Select an annotation type Annotation Bookmarks misc_feature ty3 m ty3 wy AF177870 s AF177870 stand human_T1 s human_T1 UC SOUrCE Auto annotations murine gb NC_001363 gt NC_001363 features murine gb Task view r eas 5 Sequence wy 1CF7 PDB YF murine s N
272. ouble click the sequence in the Sequence overview area or select an annotation the corresponding sequence position is made visible in the Sequence details view For a DNA sequence the Sequence details view automatically shows complement DNA strand and 6 amino translation frames T UGENE murine s NC_001363 Ay File Actions Settings Tools Window Help BOB 4A a a oa ga ca i Ot a S E S S S S E gt 2970314k 32k 33k 34k 35k 36k 37k 38k BBk 4k 44k 42k 43k 44k 45k 46k 47k 48k 3875 1125 bp 4 Toggle visibility _ loom aS 3 direct amino translations z _The original sequence 2 Ap L AGAACAACTGGACCATCCTCTAGACTGA J05 3860 J65 3870 TGTTGACCTGGTAGGAGATCTGA Value join 2970 3413 3412 3873 gt CDS CDS 3875 4999 gt E CDS CDS 5048 5203 gt P comment 0 1 a misc f See also e Navigating the Sequence details view using the Sequence overview e Selecting Amino Translation e Showing and Hiding Translations 50 Unipro UGENE Manual Version 1 20 0 Information about Sequence Context information about a sequence can be found on the Statistics tab in the Options Panel All information is contextual i e it shows statistics about the currently selected region on the selected sequence The tab includes information about e Common statistics e Length number of bases in the analyzed sequence e GC content the molar percentage of guanine and cytosine bases in an oligonucleotide sequence e
273. oved from it UGENE detects this and shows updates automatically in Project View Deleting Data To remove an object or a folder select it and press the Delete button or drag it to the Recycle bin folder All removed items are located in the Recycle bin folder 267 Unipro UGENE Manual Version 1 20 0 To delete all files from Recycle bin click on the Empty recycle bin context menu item of the Recycle bin To restore objects from the Recycle bin select them and call the Restore selected items context menu item When the database is updated outside UGENE shows these changes on your computer automatically You cannot delete any object from Recycle bin if it is opened on the other computer This situation can appear if the object was being viewed by another user when you moved it to Recycle Bin Drag n drop in the Database In the database tree you can drag n drop objects between folders folders between folders Also you can drag n drop other objects and documents from project to the database Exporting Objects from the Database The objects in the database can not be altered though they can be deleted To edit the objects you need to export them to the project then make you modifications locally and replace existing originals More detailed information about exporting you can find here 268 Unipro UGENE Manual Version 1 20 0 UGENE Public Storage UGENE provides the free to use public bioinformatics data storage This stora
274. ow the both strands are turned off 55 Unipro UGENE Manual Version 1 20 0 C c 1 m amp Jk 140k 160k i Jk 140k 160k J GATAAGA AL AT AL gt 19920 199925 199930 199 To select a translation frames use the Amino translation button and Translation frames menu Show codon table Ctrl B Translation frames k x 1 direct translation 2 direct translation 3 direct translation 1 The Standard Genetic Code 2 The Vertebrate Mitochondrial Code Selecting Sequence You can use different items from the Select submenu of the context menu to select a sequence a i K l o 7 7 o n E i 7 m E i 7 amp Goto position Ctrl 6 y F N t R E 9 R AAC 2 Select sequence region Ctl A f ITTTTTCAAACCAACCAGAACAAAGS 5 i N cae Ctrl N 1245 1250 1255 1260 1265 cite sic waa m IRABRARACTTTGGTTGCTCTTGTTTC T7 Rename item E E p iT T tT p TI Ls N KR Ss Lb 8S F L 8 i Copy Paste m m m m m m n select t TI Sequence region POEN Add Sequence between selected annotations Analyze Align Sequence around selected annotations Selecting the Sequence region context menu item opens the Select range dialog Single Range Selection Region 13 Multiple Range Selection Multi Region Here you can specify the sequence range you would like to select You can open the same dialog using the Select sequence region button on a sequence toolbar or using th
275. ow will appear here Le l of 2 selected Zero KE available Applications e To start UGENE click on the ugeneui icon You can also copy UGENE to the Applications folder by dragging it Installation on Linux e Download the appropriate version of the installation package 32 bit or 64 bit The downloaded le has tar gz extension Linux A Universal binary packages e Download 32 bit Standard or Full package Download 64 bit Standard or Full package Download 64 bit NGS package caution package size is about 4Gb e Unpack the archive You can use this command tar xf name of the downloaded tar gz file Unipro UGENE Manual Version 1 20 0 e Change the working directory to the unpacked UGENE directory cd name of the unpacked directory e Launch the UGENE GUI version using the command ugene ui or the command line version using the command ugene Several native packages for specic Linux distributions are also available UGENE is a part of Ubuntu and Fedora Linux distributions See the next chapter e Native Installation on Ubuntu e Native Installation on Fedora Native Installation on Ubuntu Ugene packages for different Ubuntu versions are available on the Personal Package Archives PPA To start installing and using software from the UGENE PPA do the following steps e Open a terminal and enter sudo add apt repository ppa iefremov ppa e Now as a one off you should tell your system to p
276. peline Explanation of the tip above Some tools are embedded into UGENE as external To be launched from the UGENE graphical interface an external tool needs a corresponding executable file The list of the external tools can be found on this page The standard package does not include the tools whereas the full package include all the required tools The NGS package besides containing the external tools contains sample data for the Cistrome pipeline hg19 genome reference genes etc so you can run it out of the box i In 2013 we worked on extending of the UGENE NGS framework with three popular pipelines for analyzing NGS data e Variant calling with SAMtools e RNA Seg data analysis with Tuxedo e ChIP Seq data analysis with Cistrome The NGS package was added as the result of this work We decided to add it as we want our users to be able to use all UGENE features out of the box However it appears that the first two pipelines are also available out of the box in the full UGENE package The work was supported by grant RUB1 31097 NO 12 from NIAID have Windows Should download installer package or portable zip bundle If you have administrative rights on Windows use the installer package It will make integration with your Windows system more tight For example it will add associations for bioinformatics formats supported by UGENE so that corresponding files are opened in it by default have Linux Which package should I use
277. ple of a task report below Task report DigestSequenceTask Finished 0 00 00 015 Digest into fragments murine gb linear Generated 10 fragments From EcoRV 138 To EcoRV 214 77 bp From EcoRV 214 To EcoRV 3227 53014 bp From EcoRV 3227 To BglII 3698 472 bp From BglII 3702 To HindIII 5023 1322 bp From HindIII 5027 To Clal 5104 78 bp To remove a notification from the Notifications popup window click the notification cross button Note that you can click on the clip button of the Notifications popup window to show the window always on top 20 Unipro UGENE Manual Version 1 20 0 Main Menu Overview Menu Description File A set of project level operations List of operations new project new document from text new workflow access remote database connect to shared database s earch NCBI genbank open open as save all save project as exp ort project close project recent files recent projects exit Actions Various actions associated with the active window List of operations go to position add copy analyze align cloning export remove edit sequence statistics for the Sequence View go to position add copy colors highlighting edit align tre e Statistics view export advanced consensus mode close active window for the Alignment Editor Settings Preferences and plugin settings Tools Various tools This menu is extended by different plugins List of ope
278. ported See this paragraph f or details about the required server configuration After that any UGENE user who knows the correct login password however can connect to the database The connected database is shown in the Project View as a document exactly the same way as if the data were located on the local computer As described in this paragraph the users can have a read only access to the database or be able to modify its content A user with a read only access can e Browse the data in the database e Open the data in the UGENE views e Export the data to the local computer Users with write access in addition can e Add new objects to the database e Create new folders to order the data in the database e Modify the folders hierarchy inside the database using drag n drop e Rename objects and folders e Delete existed objects e Delete folders All UGENE instances connected to a database constantly monitors the state of the database and shows changes made by other users UGENE accesses large remote data such as NGS assemblies so that only a viewed part of them is loaded to a client computer So if you store the assembly data on a server the data can be browsed in the UGENE Assembly Browser on a local computer almost instantly without the need to copy the data on the computer or use the hard disk space For details see the documentation below Configuring Database Connecting to a Shared Database Adding Data to the Dat
279. position weight matrices PWM s also known as position specific score matrices PSSM s The matrices came from two wide known open archives JASPAR which contains frequency matrices and UniPROBE containing weight matrices Also the Weight Matrix plugin provides a tool for creating specific position frequency and weight matrices from an existing alignment or from a file with several sequences The created matrix can be used as a profile for the search as well as the JASPAR and UNIPROBE ones To search for transcription factor binding sites in a DNA sequence select the Analyze Search TFBS with matrices context menu item The W eight matrix search dialog will appear 4 Weight Matrix Search Weight algorithm Berg and von Hippel Strands Both strands Direct strand Reverse complement strand Load list Clear results Save as annotations Results found 0 In the search dialog you must specify a file with PWM or PFM You can do so by pressing the browse button and selecting the file Also you can use the special interface to choose a JASPAR matrix by pressing the Search JASPAR database button Alternative way to specify the position weight frequency matrix is to create a specific one from an alignment or a file with several sequences with the build a new matrix tool After the profile the matrix is loaded you can adjust the threshold value The threshold sets the minimal identity score for a result to pa
280. pport of other formats is also planned so please send us a request if you re interested in a certain format To browse an assembly data in UGENE a BAM or SAM file should be imported to a UGENE database file After that you can convert the UGENE database file into a SAM file The import to a UGENE database file has both advantages and disadvantages The disadvantages are that the import may take time for a large file and there should be enough disk space to store the database file On the other hand this allows one to overview the whole assembly and navigate in it rather rapidly In addition during the import you can select contigs to be imported from the BAM SAM file So there is no need to import the whole file if you re going to work only with some contigs Note that in the future there are plans to support the other approach as well namely when a BAM SAM file is opened directly The Assembly Browser has been tested on different BAM SAM files from the 1000 Genomes Project and other sources Read the documentation below to learn more about the Assembly Browser features e Import BAM SAM File e Import ACE File e Browsing and Zooming Assembly e Opening Assembler Browser Window Assembly Browser Window Assembly Browser Window Components Reads Area Description Assembly Overview Description Ruler and Coverage Graph Description Go to Position in Assembly e Using Bookmarks for Navigation in Assembly Data e Getting Information About Read e Sho
281. pro UGENE Manual Version 1 20 0 E Hmm3 Build Effective weighting Adjust effective sequence number to achieve relative entropy target Minimum relative entropy position 0 00 Sigma parameter 45 o0 6 Use number of single linkage dusters as effective Fractional identity cutoff 0 62 O Use number of sequences as effective 6 Effective sequence number for all models to Searching Sequence Using HMM Profile HMM3 Search The HMM3 search tool reads a HMM profile from a file and searches a sequence for significantly similar sequence matches The sequence must be selected in the Project View or there must be an active Sequence View window opened If the selected sequence is nucleic and profile HMM is built from amino alignment the sequence will be automatically translated and searched in all possible frames 6 totally If a profile HMM is built for nucleic alignment the search is performed for both strands direct and complement The HMM3 search accepts the HMMER2 HMM profiles amino only as a backward compatibility feature An interesting post about using the HMMER2 models with the HMMER3 is available on the Sean Eddy s blog T HMM3 Search Acceleraton hers Query profile HMM file 7 Save annotations to Existing table P NC_014267 features NC_014267 1 gb Create new table Use auto annotations table Y Annotation parameters Group name lt auto gt sonotation type Annotation name hmm_signal Description For ex
282. quired out output file String Required type type of the matrix Boolean Optional Default false The following values are available e true dinucleic type e false mononucleic type Dinucleic matrices are more detailed while mononucleic ones are more useful for small input data sets algo algorithm used to build the matrix String Optional Default Berg and von Hippel The following values are available e Berg and von Hippel e Log odds e Match e NLG Example ugene pwm build in COI aln out result pwm Searching for TFBS with Weight Matrices Task Name pwm search Searches for transcription factor binding sites TFBS with position weight matrices PWM and saves the regions found as annotations Parameters seq semicolon separated list of input sequence files to search TFBS in String Required matrix semicolon separated list of the input PWM String Required out output Genbank file name name of the annotated regions String Optional Default misc_feature min score minimum percentage score to detect TFBS Number Optional Default 85 strand strands to search in Number Optional Default 0 The following values are available e 0 both strands e 1 direct strand e 1 complement strand 284 Unipro UGENE Manual Version 1 20 0 Example ugene pwm search seq input fa matrix Aro80 pwm Aftl pwm out res gb Building Statistic
283. r Lad The Export Image dialog appears Export to file File name ormat In the dialog you can select the image file name and its format bmp jpeg png etc For some file formats the Quality parameter also becomes available When the parameters are set click the OK button Options Panel in Assembly Browser e Navigation in Assembly Browser e Assembly Statistics e Assembly Browser Settings Navigation in Assembly Browser 153 Unipro UGENE Manual Version 1 20 0 The Navigation tab of the Options Panel in the Assembly Browser includes the list of well covered regions of the assembly and the field for searching required position WJ File Actions Settings Tools Window Help n B i i oo tiaj w Enter position in assembly 0 to 16 571 16 571 bp 8 079 to 8 143 65 bp w Most Covered Regions ES te crc 330 321 320 316 312 308 307 301 300 296 t 1 Project 8 1k 8 142C 176 1 2 3 4 5 6 7 8 9 10 4 To learn more about well covered regions refer to the Assembly Browser Window chapter To learn more about searching required position refer to the Go to Position in Assembly chapter Assembly Statistics The Assembly Statistics tab includes the following Assembly Information e Name the name of the opened assembly e Length the length of the assembly e Reads the number of reads in the assembly Also the tab can
284. r BLAST DB files base name for the BLAST database files You can see the description of the annotation saving parameters here The following advanced parameters are available 182 Unipro UGENE Manual Version 1 20 0 C Request to Local BLAST Database General options Advanced options Extension options Word size ii Gap costs 22 Filters Masks E Low complexity filter E Mask for lookup table only E Human repeats filter Mask lower case letters Word size the size of the subsequence parameter for the initiated search Gap costs costs to create and extend a gap in an alignment Increasing the Gap costs will result in alignments which decrease the number of Gaps introduced Match scores reward and penalty for matching and mismatching bases Filters filters for regions of low compositional complexity and repeat elements of the human s genome Masks for lookup table only this option masks only for purposes of constructing the lookup table used by BLAST so that no hits are found based upon low complexity sequence or repeats if repeat filter is checked Mask lower case letters with this option selected you can cut and paste a FASTA sequence in upper case characters and denote areas you would like filtered with lower case The view of the Advanced options tab depends on the selected search For the blastn search it looks like on the picture above When the bla stx search is selected in the general options
285. r each sequence opened Refer Automatic Annotations Highlighting to learn more To open the ORF Marker dialog select the Analyze Find ORFs item in the context menu 172 Unipro UGENE Manual Version 1 20 0 ORF Marker Search Settings Min length bp 100 Must terminate within region Must start with init codon E Allow overlaps Allow alternative init codons E Indude stop codon Max result 200000 ii The Bacterial and Plant Plastid Code Start codons ATG Alternative start codons TTG CTG ATT ATC ATA GTG Stop codons TAA TAG TGA 0 results found The following search settings are available Min length ORFs with length lower than Min length value will not be found Must terminate within region this option ignores boundary ORFs located beyond the search region Must start with init codon item switches the ORF Marker algorithm to the mode when any non stop amino acid code is interpreted as region start position Allow overlaps alternative downstream initiators when another start codon is located within a longer ORF i e all possible ORFs will be found not only the longest ones Allow alternative init codon option includes ORFs starting with alternative initiation codons accordingly to the current translation table Include stop codon includes stop codons into resulting annotations The other available parameters are DNA to Amino translation table defines the way start alte
286. ranovi A s Tettigonia_viridis Add 4 E Conocephalus_di Import My s Conocephalus_sp A 5 Conocephalus _p va k1 Meco Hoda CPO Lock document for editing fasta_example A Save selected documents ey Auto annotations fasta_example Bicolorana ci Aurcteacanmnotatinne facta cevarninle CO anneernh TETA j The Export Sequences as Alignment dialog will appear where you can point the result alignment file location to select a multiple alignment file format to use Genbank SOURCE tags as a name of sequences for Genbank sequences and optionally add the created document to the current project m Export Sequences as Alignment Export to file File format to use CLUSTALVY Add document to the project F Use Genbank SOURCE tags as a name of sequences for Genbank sequences only Exporting Alignment to Sequence Format Select a single object with a sequence alignment in the Project View window and click the Export Export alignment to sequence format conte xt menu item 29 Unipro UGENE Manual Version 1 20 0 Tools Window Help po41 1 g 4 R aln_example aln EEE O88 a8 Open view Add to view Unload selected objects Lock document for editing Add Import Export alignment to sequence format Export nucleic alignment to amino translation F gt Lni 3 Col1 604 Posi 6o1 B No active tasks g The Convert Alignment to Separate Sequences dialog will appear
287. rations sanger data analysis NGS data analysis BLA ST multiple sequence alignment cloning primer search for TFBS HMMER tools build dotplot generate sequence show counters e xpert discovery query designer workflow designer Window A list of active windows and basic manipulations with the windows List of operations close active view close all windows tile windows cascade windows next window previous window Help Application help and check for updates List of operations open UGENE user manual open workflow designer manual open query designer manual view UGENE documentation online visit UGENE website check for updates op en start page about Unipro UGENE Mac OS only List of operations about Unipro UGENE preferences services hide Unipro UGENE hide others show all quit Unipro UGENE The menus can be dynamically populated with new actions added by plugins Check the Plugins documentation to learn how each plugin affects global and context menus Creating New Project A project stores links to the data files cross file data associations and visualization settings Below is the description on how to create a new project manually Note that if you have no project created when opening file with a sequence an alignment or any other biological data a new anonymous project is created automatically To create a new project select the File New project menu or click the New project button on the main toolbar Th
288. rd print dialog will appear where you can select a printer to use and specify other settings 164 Unipro UGENE Manual Version 1 20 0 Extensions e Workflow Designer e DNA Annotator e DNA Flexibility e Configuring Dialog Settings e Result Annotations DNA Statistics DNA Generator ORF Marker Remote BLAST e Exporting BLAST Results to Alignment e Fetching Sequences from Remote Database e BLAST BLAST e Creating Database e Making Request to Database e Fetching Sequences from Local BLAST Database e Repeat Finder e Repeats Finding e Tandem Repeats Finding e Tandem Repeats Search Result e Restriction Analysis e Selecting Restriction Enzymes Using Custom File with Enzymes Filtering by Number of Hits Excluding Region Circular Molecule e Results e Molecular Cloning in silico e Digesting into Fragments e Creating Fragment e Constructing Molecule e Available Fragments Fragments of the New Molecule Changing Fragments Order in the New Molecule Removing Fragment from the New Molecule Editing Fragment Overhangs Reverse Complement a Fragment Other Constuction Options e Output e Creating PCR Product e In Silico PCR e Primers Details e Primer Library e Secondary Structure Prediction e SITECON e SITECON Searching Transcription Factors Binding Sites e Types of SITECON Models e Eukaryotic e Prokaryotic e Building SITECON Model e Smith Waterman Search e HMM2 e Building HMM Model HMM Build e Calibrating HMM Model HMM Calibrate e Sear
289. re highlighting Annotations highlighting Search in Sequence To search for a pattern s in a sequence go to the Search in Sequence tab of the the Options Panel in the Sequence View Input the value you want to search in the text field and click the Search button To search multiple patterns input the patterns separated by a new line in the pattern text field To add a new line symbol Ctrl Enter may be used You can input the value as sequence or name of the sequence in the FASTA format and sequence after that Fo Search in Sequence s ga Search for ACG By default misc_feature annotations are created for regions that exactly match the pattern Find below the description of the available settings e Load Patterns from File 58 Unipro UGENE Manual Version 1 20 0 e Search Algorithm e Search in e Other Settings e Annotations Settings Load Patterns from File Load patterns from file Path amm Use this checkbox to load patterns from file When this option is active the Search for field is disabled Search Algorithm Search algorithm Algorithm Exact This group specifies the algorithm that should be used to search for a pattern The algorithm can be one of the following e nsDel there could be insertions and or deletions i e a pattern and the searched region can vary in their length You can specify the percentage of the pattern and a searched region match i
290. re available e fasta e fastq e genbank e off e raw 274 Unipro UGENE Manual Version 1 20 0 Example ugene convert seq in human_Tl fa out human_Tl gbk format genbank Converting MSA Task Name convert msa Converts a multiple sequence alignment file from one format to another Parameters in input multiple sequence alignment file String Required out name of the output file String Required format format of the output file String Optional The following values are available e clustal default e fasta e mega e msf nexus e phylip interleaved e phylip sequential e stockholm Example ugene convert msa in CBS sto out CBS format msf Extracting Sequence Task Name extract sequence Extracts annotated regions from an input sequence Parameters in semicolon separated list of input files String Required out output file String Required annotation names list of annotations names which will be accepted or filtered String Optional annotation names file file with annotation names separated with whitespaces which will be accepted or filtered String Optional accumulate accumulate all incoming data in one file or create separate files for each input In the latter case an incremental numerical suffix is added to the file name using True by default Boolean accept or filter if set to true accepts only the specified annotations if set to fals
291. rently supported chromatogram file formats are ABIF and SCF To view a chromatogram just open an interesting file in UGENE by standard means e g drag amp drop the file or press the Ctrl O shortcut The Chromatogram Viewer is automatically embedded into the generic Sequence View if chromatogram data are found as on the screenshot below 98 Unipro UGENE Manual Version 1 20 0 dP Aixberezikov dna m 1 50 100 150 200 20 300 350 400 450 500 220 600 650 T T90 800 650 Chromatogram view zoom in to see base calls oToGOCOA il li i dh iia 233 260 280 300 320 340 360 360 400 420 440 460 480 500 520 540 560 560 600 620 640 660 660 704 L gl After zooming in more chromatogram details are available AATAT TGAACG TAG GTGCGATAAATAAG 0 0 0 6 0 0 0 6 6 0 0 0 6 6 6 0 0 6 0 0 0 0 0 6 0 0 AAT AT TG A A cG T AG GT G Cc G AT A AAT AA SE bo A C G MiA IER FNR SA Aila To edit a sequence data right click on the chromatogram view and select the Edit new sequence item in the appeared context menu The following dialog will appear m Add New Document Document format Document location E Compress file Select new document format and location and click on the Create button The original DNA sequence is not allowed to be changed however you can add and modify a new sequence stored in a separate file The sequence being edited is displayed right above the original o
292. ress the Build button to run the analysis with the parameters selected and build a consensus tree PhyML Maximum Likelihood The Building Phylogenetic Tree dialog for the PhyML Maximum Likelihood method has the following view Build Phylogenetic Tree Tree building method PhyML Maximum Likelihood Substitution Model Branch Support Display Options Substitution model HKYs5 Y Equilibrium frequencies optimized empiri F Transition transversion ratio Oo Proportion of invariable sites 0 00 Number of substitution rate categories E Gamma shape parameter save tree to Remember Settings Restore Default There following parameters are available Substitution model parameters selection of the Markov model of substitution Substitution model model of substitution Equilibrium frequencies equilibrium frequencies Transition transversion ratio fix or estimate the transition transversion ratio in the maximum likelihood framework Proportion of invariable sites the proportion of invariable sites i e the expected frequency of sites that do not evolve can be 139 Unipro UGENE Manual Version 1 20 0 fixed or estimated Number of substitution rate categories number of substitution rate categories Gamma shape parameter the shape of the gamma distribution determines the range of rate variation across sites Branch support parameters selection of the method that is used to measure branch support Use fast
293. rev alginment 1 5 Mask level c 0 50 E Prefer hard dipping in SAM output H NOTE bwa sw performs alignment of long sequencing reads Sanger or 45 4 It accepts reads only in FASTA or FASTQ format Reads should be compiled into single file There are the following parameters Reference sequence DNA sequence to align short reads to This parameter is required Result file name file in SAM format to write the result of the alignment into This parameter is required SAM output always save the output file in the SAM format the option is disabled for BWA Short reads each added short read is a small DNA sequence file At least one read should be added You can also configure other parameters 233 Unipro UGENE Manual Version 1 20 0 Index algorithm a algorithm for constructing BWA SW index It implements three different algorithms e is designed for short reads up to 200bp with low error rate lt 3 It does gapped global alignment w r t reads supports paired end reads and is one of the fastest short read alignment algorithms to date while also visiting suboptimal hits e bwtsw is designed for long reads with more errors It performs heuristic Smith Waterman like alignment to find high scoring local hits Algorithm implemented in BWA SW On low error short queries BWA SW is slower and less accurate than the s algorithm but on long reads it is better e div does not work for l
294. rformance than BWA backtrack for 70 100bp Illumina reads Open Tools Align to reference submenu of the main menu r T UGENE human_T1 s human_T1 UCSC April 2002 chr7 115977709 117855134 File Actions Settings Window Help E E F E A DNA assembly ae cs Align to reference Agn ees as fa HMMER tools Build index d ag a Multiple alignment aE q Stes BLAST Et CVU55762 gb 20k 30k 40k 30k IE Ah Generate sequence dP a cvu55762 Select the Align short reads item to align short reads to a DNA sequence using BWA MEM Or select the Build index item to build an index for a DNA sequence which can be used to optimize aligning of short reads e Aligning Short Reads with BWA MEM e Building Index for BWA MEM Aligning Short Reads with BWA MEM When you select the Tools Align to reference Align short reads item in the main menu the Align Sequencing Reads dialog appears Set 235 Unipro UGENE Manual Version 1 20 0 value of the Align short reads method parameter to BWA MEM The dialog looks as follows m Align Sequencing Reads Alignment method Reference sequence Result fle name Library Single end SAM output Short reads Base Options Index algorithm a Score for a match A Number of threads t Mismatch penalty 8 Min seed length 4 Gap open penalty 0 Band width w Gap extention penalty Dropoff d 100 Penalty for clipping 4 Internal seeds length 1
295. rithm to calculate the repeats 108 Unipro UGENE Manual Version 1 20 0 e Auto e Suffix index e Diagonals i The specified algorithm is provided to the Repeat Finder plugin as an input parameter In most cases the Auto value is appropriate Minimum repeat length allows to draw only such matches between the sequences that are continuous and long enough For example if it equals to 3bp then only repeats will be found that contain 3 and more base symbols Press the 7k button to automatically adjust the Minimum repeat length value Such value will be set that there will be about 1000 repeats found Repeats identity specifies the percents of the repeats identity Press the 700 button to set the 100 identity After the parameters are set press the OK button The dotplot will appear in the Sequence View 1 20k 40k 60K GOK 100k 120k 140 426 z _ a Sor nyt Oey So oe ieee en OS eee ba er ee he as Lo ee eet a Ta Tr ae e anaes T T CSTR e g er ae eile Oea ye eap Ae Ho e ke dp a Egien E Pee ai ong tere od ee e E pO EE aaia a Oe ma PO aod G pen E Be 3 5 c 1 sat pacer oo lee eee cone re soe oo Oe ae Ea E Oo ose ee r pek i ee wy a SUE pang eee ig BTU ELA a Be Rea Seek a AWN a Oe Mie ae te Cota ME te eo Oe ee Eel on ne Pa Joa pppoe gP oa tages se o amta tee er i ta ds T a we a4 t Trei in an ay Sr TOK BD Rati Pb pe dea ded ds TA a aU a eee te SE eae Oe ap A i E Taara yyt Fi T E A a E aa LELE ana UDAR
296. rnative start and stop codons are encoded Strand where to search the ORFs in the direct strand in the complement strand or in both strands Preview allow to preview the regions strands and lengths of the found ORFs 173 Unipro UGENE Manual Version 1 20 0 Clear results becomes available when some results have been found clears these results To set the saving parameters go to the Output tab of the dialog Here you can modify the annotations saving parameters Group name Description and a file to save the annotation to Results When the search parameters has been selected and the OK button has been pressed in the dialog the auto annotating becomes enabled In the Annotations editor the ORFs annotations can be found in the Auto annotations orf group After the search has been finished you can browse the results sort them by length strand or start position and save as annotations to the original sequence in the Genbank format For more information about codons use the codon table It depends on the translation code selected for the sequence To show or hide the table use Ctrl T shortcut or click the Show codon table submenu of the Amino translation toolbar button menu _ Ie GI g b F o Show codon table Ctrl T Translation frames b 1 The Standard Genetic Code l 2 The Vertebrate Mitochondrial Code 3 The Yeast Mitochondrial Code 4 The Mold Protozoan and Coelenterate Mitochondria and the Mycoplasm
297. romatograms Simultaneously To add another sequence to the Sequence View drag the required sequence object from the Project View and drop it in the Sequence View area Note that the dragged object is the sequence object not the chromatogram object ate 90 JRI O7_copy sequence 110 20 a0 40 50 Chromat 4 O0 JRI O7 scf s 90 JRI 07 sequence bee cy c 90 JRI 07 chromatogram af 90 JR1 07_copy scf _ a s 90 JRI 07 copy sequence i 1 10 20 aj 40 S50 E The result will look like this 90 JRI 07 sequence WIO BR mihi ce sia A a vt Chromatogram view zoom in to see base calls oOTOGOoCoA 1 m I III 1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 213 4 90 JRI 07_copy sequence em O B T E aja aaga D a o o a o aia aa aa aia ia ia aa ia Chromatogram view zoom in to see base calls are oToGoCoA iil u an AN aa a a e m AA a ann dn 120 13N 140 15N 16N 17N 1AM 1a 2N 2 You can also use the Lock scales and Adjust scales global actions for the chromatograms For example if you lock the scales you are able to scroll the sequences simultaneously Also when you select a sequence region in one sequence the same region is selected in the second sequence 101 Unipro UGENE Manual Version 1 20 0 U New Project UGENE 90 JRI O 7_copy 90 JRI O7_copy sequence U Fie Actions Settings Tools Window Help ReBiAaAz e abakan 1 06
298. rt Reads Vizualization e Reads Highlighting e Reads Shadowing Associating Reference Sequence Associating Variations Consensus Sequence Exporting e Exporting Reads Exporting Visible Reads Exporting Coverage Exporting Consensus Exporting Consensus Variations e Exporting Assembly as Image e Options Panel in Assembly Browser e Navigation in Assembly Browser e Assembly Statistics e Assembly Browser Settings e Assembly Browser Hotkeys e Assembly Overview Hotkeys e Reads Area Hotkeys Import BAM SAM File To start working with an assembly import it to the UGENE database file To do this open the assembly file For assembly file without header you need to choose a referenece sequence 141 Unipro UGENE Manual Version 1 20 0 T Import SAM File Source URL C work ugene data samples Assembly chrM sam Reference The SAM file does not contain the header Please choose the reference sequence Import unmapped reads Destination URL C work ugene data samples Assembly chrM sam ugenedb Select the referense sequence and click mport button For other assembly files the following dialog appears T Import BAM File Source URL C work ugene data samples Assembly chrM sorted bam Assembly name Length 1 chrM 16 5 1 Import unmapped reads Destination URL The Source URL field in the dialog specifies the file to import The nfo button nearby can be used to obtain additional information about the file There
299. s A Rulers No active tasks gy E The Export Selected Sequence Region dialog will appear which is similar to the Export Selected Sequences dialog described here Exporting Sequence of Selected Annotations Open the Sequence View with document that contains annotations A good candidate here could be any file in Genbank format with both sequence and annotations Select a single or several annotations or annotation groups in the Annotation editor click the right mouse button to open the context menu and select the Export Export sequence of selected annotations item 64 Unipro UGENE Manual Version 1 20 0 Invert annotation selection Rename item F2 Paste annotations Ctrl Shift V Go to position Ctrl G Select sequence region Ctr A New annotation Ctrl N J 1 Project Copy Paste Select Add Analyze brez sii 1065 1070 Cloning rCTGACAATGGTGAGGGAATTG Fetch sequences from remote database Export Edit Export selected sequence region _ Export sequence of selected annotations Remove Export annotations join 2970 3413 3412 38 3 gt B CDS 3875 4999 gt re comment 0 1 mise feature M No active tasks yg The Export Sequence of Selected Annotations dialog will appear which is similar to the Export Selected Sequences dialog described here Locking and Synchronize Ranges of Several Sequences An important feature of the Sequence zoom view is the ability to synchro
300. s M Lern pdb A Wy e 1CRN chain 1 sequence a 1CRN chain i annotation 3d 1CRN M 1FDL pdb Dy s 1FDL chain 1 sequence e Press the Add button on the toolbar The Select Item dialog will appear Select 8d objects to add Hint Use the Ctrl keyboard button to select several objects hy Select Item 4 Bice sh 3d 1CF7 4 1CRN PDB Below you can see the 3D Structure Viewer with two views 97 Unipro UGENE Manual Version 1 20 0 4 3D Structure Viewer Active view 2 1cRN Cc H q F Display Links Add amp To select an active view click on the view area or select an appropriate value in the Active view combo box on the toolbar To synchronize the views press the Synchronize 3D Structure Views sticky button on the toolbar see the image above When the button has been pressed the 3D structures are moved zoomed and spinned synchronously Press the button again to stop the views synchronization The views that are no more required can be closed by selecting the Close button in the 3D Structure Viewer context menu Also you can hide show views for a while Use the menu of the green arrow button on the toolbar to do it w Show 1CRN w Show 1V6c w Show IMOT Close 3D Structure Viewer L Notice that the 3D Structure Viewer can be closed from this menu Chromatogram Viewer The Chromatogram Viewer plugin brings DNA chromatogram data viewing and editing capabilities into UGENE Cur
301. s are encoded as characters A C G T A blue C green G orange T red UGENE Genome Aligner The UGENE Genome Aligner is a fast short read aligner It aligns DNA sequences of various lengths to the reference genome with configurable mismatch rate It is available from the Tools DNA assembly submenu of the main menu Ee Window Help DNA assembly a Align short reads Test runner zi Build index amp SITECON b pn i Convert UGENE Assembly data base to SAM format Select the Align short reads item to align short reads to a DNA sequence or Build index item to build an index for a DNA sequence which can be used to optimize aligning short reads to the sequence e Aligning Short Reads with UGENE Genome Aligner e Building Index for UGENE Genome Aligner e Converting UGENE Assembly Database to SAM Format Aligning Short Reads with UGENE Genome Aligner When you select the Tools DNA Assembly Align short reads item in the main menu the Align Short Reads dialog appears Set the Align short reads method parameter to UGENE Genome Aligner The dialog looks as follows 238 Unipro UGENE Manual Version 1 20 0 T Align Sequencing Reads Alignment method Reference sequence Result file name Library Single end E Prebuilt index W SAM output Short reads Order Common parameters E Mismatches allowed Mismatches number Percentage of mismatches Align options C Use GPU optimization E Align reverse co
302. s marked with the lock icon It means that the storage provides the read only access Any data modifications are unavailable for such type of access importing removing or replacing of data Each genome folder contains the NFO text object It is the information about the genome or its source You can export the data to your computer for working with the data locally There are hundreds of plasmids in the storage Use the name filter for fast navigating and searching an interesting plasmid Project x Objects Ay s AF058756_Cloning vector pFR Luc compl a AFO58756_Clo ning vector pFR Luc compl The list of available genomes Human hg19 Mouse mm9 Arabidopsis thaliana TAIR 10 C elegans ce6 Drosophila melanogaster dm3 Escherichia coli str K 12 substr MG1655 K12 NC_000913 3 Human Immunodeficiency Virus HIV 2 Mycobacterium tuberculosis NC_000962 3 Salmonella Enterica NC_016856 1 Vibrio cholerae NC_002505 Yeast Saccharomyces cerevisiae sacCers Zebrafish Danio rerio danRer7 2 0 Unipro UGENE Manual Version 1 20 0 UGENE Command Line Interface UGENE command line interface CLI was developed keeping in mind the following principles e To make it as easy as popular shell commands e To include all significant UGENE features e To allow users to add their own commands To use UGENE CLI make sure to add the path to the UGENE executable to your PATH environment variable Th
303. s produce the same results as classic but much faster To use these optimizations our system must support these capabilities OPENCL SSE2 or SW_classic Scoring matrix scoring matrix Gap open penalty penalty for opening a gap Gap extension penalty penalty for extending a gap Output settings settings of the otput file Working with Sequences List Adding New Sequences Copying Sequences Renaming Sequences Sorting Sequences Shifting Sequences Collapsing Rows Adding New Sequences You can add new sequences to an alignment using the Add submenu in the Actions main menu or the context menu There are two ways to add a new sequence to the current alignment e From a file in the compatible format FASTA GenBank etc The list of the supported data formats can be found here e From the current project If you activate this item the following dialog will appear Select Item a H 1CF7 PDB Py s 1CF chain 3 sequence A s 1CF7 chain 4 sequence You will see the Project View tree filtered to show only appropriate sequences Select the items to add and press the Ok button 129 Unipro UGENE Manual Version 1 20 0 Copying Sequences To copy current selection click the Copy Copy selection item in the Actions main menu or the context menu The hotkey for this action is Ctrl C To copy one or several sequences do the following e Select the sequences in the Sequence list area e Select the Copy Copy selection co
304. s the left arrow to search in the direction From right to left from bottom to top If the pattern is found the result will be focused and highlighted in the Sequence area You can continue the search in any direction from this position Consensus Each base of a consensus sequence is calculated as a function of the corresponding column bases There are different methods to calculate the consensus Each method reveals unique biological properties of the aligned sequences The Alignment Editor allows switching between different consensus modes To switch the consensus mode go to the General tab of the Options Panel or activate the context menu using the right mouse button or the Actions menu and select the Consensus mode item and General tab will be opened automatically w Reference sequence Select and add w Alignment info Length Sequence number Y Consensus mode Consensus type Default There are several consensus modes e JalView Default it is based on the JalView algorithm Returns if there are 2 characters with high frequency Returns symbol in lower case if the symbol content in a row is lower than the specified threshold e ClustalW emulates the ClustalW program and file format behavior e Levitsky this algorithm is proposed by Victor Levitsky to calculate consensus of DNA alignments At first it collects global alignment frequencies for every symbol using extended 15 symbols DNA alphabet Then
305. sequences to the alignment To do it select the sequence object s in the Project View an d click the Align sequence to this alignment toolbar button The sequence s will be aligned to the alignment automatically Pairwise Alignment To align two sequences go to the Pairwise Alignment tab of the Options Panel Pairwise Alignment wY Sequences Select and add Select and add Algorithm Algorithm settings Gap open penalty 217 00 Gap extension penalty 39 00 Terminate gap penalty 39 00 z Bonus score 283 00 wY Output settings In new window Output file a PairwiseAlignmentResult aln e Align Select two sequence from the original alignment select the parameters and click on the Align button The following parameters are available 128 Unipro UGENE Manual Version 1 20 0 Algorithm algorithm of the pairwise alignment There are two algorithms Hirschberg KAlign algorithm has the following parameters Gap open penalty indicates the penalty applied for opening a gap The penalty must be negative Gap extension penalty indicates the penalty applied for extending a gap Terminate gap penalty the penalty to extend gaps from the N C terminal of protein or 5 3 terminal of nucleotide sequences Bonus score a bonus score that is added to each pair of aligned residues Smith Waterman the following parameters are available Algorithm version version of the algorithm implementation Non classic version
306. should be more than 20 Similarity score of an overlap parameters The following parameters are used to calculate the similarity score of an overlapping alignment Match score factor m a match at bases of quality values q1 and q2 is given a score of m min q1 q2 where m is the specified value The specified value should be more than 0 Mismatch score factor n a mismatch at bases of quality values q1 and q2 is given a score of n min q1 q2 where n is the specified value The specified value should be less than 0 Gap penalty factor g a base of quality value q1 in a gap is given a score g min q1 q2 where g is the specified value q2 is the quality value of the base in the other sequence right before the gap The specified value should be more than 0 The similarity score is caclulated as the sum of scores of each match each mismatch and each gap Based on this value and the following value some overlaps are removed 242 Unipro UGENE Manual Version 1 20 0 Overlap similarity score cutoff s remove overlaps with similarity scores less than the specified value The specified value should be more than 250 Length and percent identity of an overlap parameters Overlap length cutoff o minimum length of an overlap in base pairs The specified value should be more than 15 base pairs Overlap percent identity cutoff p minimum percent identity of an overlap The specified value should be more tha
307. sion 1 20 0 Y Forward primer CACACGTACTOACAGTCAGCATACGK Tm 61 39 C 28 mer C 4h Mismatches 7 bp The following dialog will appear U Choose Primer GL content 76 Length bp Sequence AAAAAACGT The table consists of the following columns name GC content Tm Length bp and sequence Select primer in the table and click the C hoose button Click the Reverse complement button for making a primer Sequence reverse complement In Silico PCR Y Forward primer ACACACGTACTGACAGTCAGCATACG Tm 61 39 C aos ri Click Show primers details for seeing statistic details about primers When you run the process the predicted PCR products appear in the products table Products table There are three columns in the table e region of product in the sequence e product length e preferred annealing temperature Click the product for navigating to its region in the sequence Click the Extract product s button for exporting a product s in a file or use double click for that 199 Unipro UGENE Manual Version 1 20 0 Region Length Ta 60622 63999 31 6 58 56 Extract product s e Primers Details e Primer Library Primers Details Click Show primers details for seeing statistic details about primers 4 Mismatches 10 bp w Settings Maximum product 5000 bp Show primers details pr The following dialog will appear 200 Unipro UGENE Manual Version 1 20 0 RPinenoe Se eel
308. sponding restriction sites in the sequence Using Custom File with Enzymes To load a custom file with enzymes click the Enzymes file button and browse for the file The file must be of the Bairoch format For details about the format refer http rebase neb com rebase rebase f19 html To export enzymes use the Export enzymes button You can also save the currently selected enzymes to a file and load saved selection Click the Save selection and Load selection buttons correspondingly to do it Filtering by Number of Hits To filter the results by the number of restriction sites found for an enzyme check the Filter by number of results check box and input the minimum value and the maximum value of hits Excluding Region To exclude a sequence region from the search check the Exclude region check box and input the start and the end positions of the region If a subsequence has been selected before opening the dialog you can click the Selected button to automatically fill the values with the selected subsequence s start and end positions Circular Molecule To consider the sequence as circular and be able to search for restriction sites between the end and the beginning of the sequence check the Circular molecule option Example Let s consider e The sequence is CTGC CAC e Aarl restriction enzyme with recognition sequence CACCT GC has been checked In this case if the Circular molecule option has been checked the restrict
309. ss The more the result score is the more it is homologically related to the aligned region By changing the threshold you can filter low scoring results If the loaded matrix is a position frequency matrix you must also specify the algorithm to build the corresponding position weight matrix which will represent the transcription factor There are four algorithms available 245 Unipro UGENE Manual Version 1 20 0 Weight algorithm Berg and von Hippel Berg and von Hippel Log odds Strands F a a Also you can add a selected matrix with the specified Minimal score and the Algorithm to the matrices list To do it select the matrix and other options and press the Add to queue button The plugin will search with all matrices specified in the list You can use the Save list button to export the list of matrices to a csv file Later the list can be loaded from the file using the Load list button The rest options are standard sequence search options the strand and the sequence region where to search for matches After specifying the necessary options press the Search button The found results will appear in the dialog table The corresponding results identity scores are in the Score column Range Matrix Strand Score ve 199944 199949 MADZ71 1 pfm Direct strand 31 26 f 199943 199948 MADZ71 1 pfm Direct strand 62 59 199942 199947 MADZ71 1 pfm Direct strand Dd De Ye 199941 199946 Made 71 1 pFm Direct strand 26 06 199940
310. t 26 855 27k af 2k 24 5k gt To deselect the repeat either click on other repeat or hold Ctrl and click somewhere on the dotplot Interpreting Dotplot Identifying Matches Mutations Invertions etc Using a dotplot graphic you can identify such the following differences between the sequences 1 Matches A match between sequences looks like a diagonal line on the dotplot graphic representing the continuous match or repeat 2 Frame shifts a Mutations Mutations are distinctions between sequences On the graphic they are represented by gaps in diagonal lines They interrupt matches b Insertions Insertions are parts of one sequence that are missed in the another while the surrounding parts match In other words an insertion is a subsequence that was inserted into a sequence Graphically insertions are represented by gaps which lie only on one axis A little shift towards the other axis indicates a mutation involved c Deletions A deletion is a subsequence that was deleted from a sequence A deletion from sequence A found in sequence B can be considered as an insertion into sequence B and contained in sequence A 111 Unipro UGENE Manual Version 1 20 0 1 10k 20K 30k 40k 50k 60k FOK GOK 90k 100k 110k 120k 140 426 Aly aS ares ca Deore eee ata T i fr 4 li Ber ate Sa i D J hes ae eee Te a en als mag nAi av mas y Ai PAE pai g eee oe ae nae r oo LE iea TE EEE ao a8 Iip mi a A nnn 1
311. t e Navigating in Dotplot 84 Unipro UGENE Manual Version 1 20 0 Zooming to Selected Region Selecting Repeat Interpreting Dotplot Identifying Matches Mutations Invertions etc Editing Parameters Filtering Results Saving Dotplot as Image Saving and Loading Dotplot Building Dotplot for Currently Opened Sequence Comparing Several Dotplots Circular Viewer The Circular Viewer plugin provides capability to show the circular view of a nucleotide sequence Usage example Open a nucleotide sequence object in the Sequence View The Show circular view button is available on the sequence toolbar cVvu55762 dna Of Show circular view 1 300 1k 1 5k 2k 2 5k File Actions Settings Tools Window Help re er TE amp amp Eo 1 Project EBV_rev_primer Restriction Sites Map Te gt 7 BamHI 0 sites ee a N AOE 1 E Bbsl 0 sites in BglII 0 sites Clal 0 sites Dral 0 sites EcoRI 0 sites HindIll 0 sites PstI 0 sites Sall 0 sites Smal 0 sites Xmal 0 sites X eo Th FH FT TF a E I 3242 3244 3246 3248 3250 3252 3254 3256 3258 3260 3262 3264 3266 3268 3270 3273 GAAAAC CT CT GCGACA CAT GCA GE TCC CEG AGC AC Y A E M Q 240 245 250 255 260 265 270 280 305 310 315 320 AAGGCGGGTAAGAGGCGGGGTACCGACTGATTAAAAAAAATAAATACGTCTCCGGCTCCGGCGGAGCCGGAGACTCGATAA A Name Value gt AF013258 features pSG5 gb gt Auto annotations pSG5 gb AF01
312. t the alignment of unrelated segments using 0 by default Number gap open penalty Gap Open Penalty Must be negative best matches get a score of 1000 using 50 by default Number iter max Number of iteration on the progressive alignment 0 no iteration default 1 Nseq iterations using 0 by default Number toolpath T Coffee location using the path specified in UGENE by default String tmpdir Directory to store temporary files using UGENE temporary directory by default String in Input alignment Url datasets format Document format of output alignment using clustal by default String out Output alignment String Example 282 Unipro UGENE Manual Version 1 20 0 ugene align tcoffee in test aln out test_out aln format clustal Building PFM Task Name pfm build Builds a position frequency matrix from a multiple sequence alignment file Parameters in semicolon separated list of input MSA files String Required out output file String Required type type of the matrix Boolean Optional Default false The following values are available e true dinucleic type e false mononucleic type Dinucleic matrices are more detailed while mononucleic ones are more useful for small input data sets Example ugene pfm build in COI aln out result pfim Searching for TFBS with PFM Task Name pfm search Searches for transcription factor binding sites TFB
313. ta folder data custom_annotations plasmid_features txt ClustalO Clustal is a widely used multiple sequence alignment program It is used for both nucleotide and protein sequences Clustal Omega is the latest addition to the Clustal family It offers a significant increase in scalability over previous versions allowing hundreds of thousands of sequences to be aligned in only a few hours It will also make use of multiple processors where present Clustal home page hitp www clustal org If you are using Windows OS there are no additional configuration steps required as Clusta O executable file is included to the UGENE distribution package Otherwise e Install the Clustal program on your system e Set the path to the ClustalW executable on the External tools tab of UGENE Application Settings dialog Now you are able to use ClustaO from UGENE Open a multiple sequence alignment file and select the Align with ClustalO item in the context menu or in the Actions main menu The Align with ClustalO dialog will appear see below where you can adjust the following parameters Number of iterations number of Combined guide tree HMM iterations Max number guidetree iterations maximum guide tree iterations Max number of HMM iterations maximum number of HMM iterations Number of CPUs being used number of processors to use Set options automatically set options automatically might overwrite some of your options 256
314. tall executable By default the path specified in the Application Settings is applied String Optional Default default 2 7 Unipro UGENE Manual Version 1 20 0 tmpdir directory for temporary files By default the path specified in the Application Settings is applied String Optional Default default in semicolon separated list of input sequence files String Required dbpath path to the BLAST database files String Required dbname base name of the BLAST database files String Required out output Genbank file the results of the search are stored as annotations String Required name name of the annotations String Optional Default blast result p type of the BLAST search String Optional Default blastn The following values are available e blastn e blastp e blastx e tblastn e tblastx e expectation value threshold Number Optional Default 10 Example ugene local blast in input fa dbpath dbname mydb out output gb Local BLAST Search Task Name local blast Performs a search on a local BLAST database using BLAST Q BLAST is used as an external tool and must be installed on your system Parameters toolpath path to an appropriate BLAST executable e g blastn blastp etc By default the path specified in the Application Settings is applied String Optional Default default tmpdir directory for temporary
315. tasks y 2 The hotkey for this action is Alt 3 It is possible to configure the Log View settings the level of the log to show ERROR INFO DETAILS TRACE the category Algrorithms Tasks etc and the format of the log messages format of the dates etc This settings can be configured in the UGENE Application Settings Notifications The Notifications component shows notifications for tasks reports seattle aa l Notifications INFOJ 15 54 Converting assembly from Klebsislla sort bam to Klebsislla DETAILS 16 54 Importing assembly pkF70 1 of 3 DETAILS 16 55 Succesfully imported 136066 reads for assembly pkF 7d fey 16 27 31 Report for task X DETAILS 16 55 Importing assembly pkf140 2 of 3 DETAILS 16 57 Succestfully imported 416287 reads for assembly pkf14 DETAILS 16 57 Importing assembly pKF94 3 of 3 INFO 17 01 Canceling task Convert BAM to UGENE database Klebsisl INFO 17 01 Canceling task BAM SAM file import Klebsislla sort bam INFO 17 01 Canceling task Convert BAM to UGENE database Klebsisll MHEIC F lL Fs i ack Adci klebsiela en CUO ar mer ha oredect KI i If a task has finished without errors the notification is blue If an error has occured during the task execution the notification is red If a warning has occured during the task execution the notification is yellow To open a task report click on the corresponding notification See an exam
316. tation context menu item UGENE sars s NC_004718 File Actions Settings Tools Window Help o BH A a Sina fs et DB dP Nc_004718 dna Goto position Ctrl G Select sequence region Ctrl A A New annotation Rename item Copy Paste r a Analyze Objects with annotations Align Qualifier Cloning Fetch sequences from remote database Export Edit Narne Auto annotations sars gb 4 NC 004718 features sars q b gt eo UTR 0 1 Remove a IAR OF Al Rulers a cos 0 14 a E CDS B Disable CDS highlighting codon_start h yrei This will activate a dialog where to set up annotation parameters 69 Unipro UGENE Manual Version 1 20 0 hy Create Annotation Group name source Glycosylation Site Homeodomain Annotation name iDNA P Insertion as Location Intron J Region B ak format J 5egment Leucine Zipper Domain Loci Complement LTR Mature Peptide 0 GenBank EMBL format Misc Binding Site Misc Difference Misc Feature Existing table 9 NC_001363 features murine gb C Create new table Use auto annotations table The dialog asks where to save the annotation It could be either an existing annotation table object a new annotation table or auto annotations table if it is available You can also specify the name of the group and the name of the annotation If the group name is set to lt auto gt UGENE will use the group name as the name for
317. tation s to Existing table 9 NC_014267 features NC_014267 1 9b O Create new table Use auto annotations table Y Annotaton parameters Group name lt auto gt Description oh Search timeout 10min General options are Select the search type in the remote databases the blastn search is used for nucleotide sequences blastp and cdd searches are used for amino sequences UGENE also provides a way to use blastp and cdd searches for nucleotide sequences This is achieved by translating the nucleotide sequence into the amino sequences When a sequence is translated the translation table from the active Sequence View is used Finally all 6 translations are used to query the remote database with the selected blastp or cdd search Expectation value this option specifies the statistical significance threshold for reporting matches against database sequences Lower expect thresholds are more stringent leading to fewer chance matches being reported Max hits the maximum number of hits that will be shown not equal to number of annotations The maximum availablle number is 5000 Database the target database Search for short nearly exact matches automatically adjusts the word size and other parameters to improve results for short queries Megablast select this option to compare query with closely related sequences It works best if the target percent identity is 95 or more but it is very fast You ca
318. terested in Negative sequence base doesn t have it You also may generate negative sequences automatically ExpertDiscovery will extract complex signals which reflect a structure of your regulation object The more sequences you provide the better will be the result Positive sequences file E Generate negative sequences Negatives per positive amount 100 Negative sequences file Load the sequences you want to analyze by choosing any file with a sequence or multiple sequences Positive sequence base contains a regulation object you are interested in Negative sequence base doesn t have it You also may generate negative sequences automatically ExpertDiscovery will extract complex signals which reflect a structure of your regulation object The more sequences you provide the better will be result Click on the Next button The following dialog will appear m Positive and Negative Sequences Markup On this step you need to load markups for the sequences Markaup is an annotation of a sequence with elementary signals Markup gives information where elementary signals are located in the sequences Complex signals will be build from the elementary signals and operations applied to them Load markup for your sequences in specified XML format or genbank format Nucdeotides Markup Positive sequences markup file Negative sequences markup file Append to Current Markup Here you can to load markups
319. the group You can use the characters in this field as a group name separator to create subgroups If the annotation name is set to by type UGENE will use the annotation type from the Annotation type table as the name for the annotation Also you can add a description in the corresponding text field The Location field contains annotation coordinates The coordinates must be provided in the Genbank or EMBL file formats If you want to annotate complement strand sequence check the corresponding checkbox for the simple format or surround the coordinates with the complement word or press the last button in the corresponding row to do it automatically Note that by default the Location field contains the coordinates of the selected sequence region Once the Create button is pressed the annotation is created and highlighted both in the Sequence overview and the Sequence details viewa reas H E D T R E W Q z L z N A T ie 4 6 8 10 12 14 16 18 20 22 34 26 28 30 32 34 36 38 40 42 44 46 49 ITTACTITCTGGSSTGCEGSGCATCCACCETICGATCGCAATTICATTGCGESMISG I E m gt V oR L H C A L E L L w Mame gt value G 9 Annotations My Document gb B misc_feature 0 1 O misc_feature 4 09 E MC 001363 Features NT _001363 gb H gD cos 0 t Selecting Annotations 70 Unipro UGENE Manual Version 1 20 0 To select one annotation click on it To select several annotations hold Ctrl key while clicking on the annotati
320. tide sequence using different algorithms Repeats finder ORF finder Weight matrix matching etc at the same time imposing constraints on the positional relationship of the results obtained from the algorithms A user friendly interface is used to create a schema of the algorithms and constraints U My Project UGENE File Actions Settings Tools Window Help ell B ta Eo b ca View mode Property Editor a B e A CDD ORF Finds Open i Reading Frames ORFs in supplied nucleotide sequence stores Found regions as annotations I ORF i Primer representing algorithms Frotein sequences are ga skipped if any a Repeats ORDF are M sequence ae Restriction site regions that cous potentat encode a ACG patt protei and usualy gire E aem a good indication of fhe presence of a gene in ihe w S Sitecon Parameters SW Smith Waterme Name Value i Allow al e codons False Weight matrix Require stop codon False Constraints Annotate as ORF Max length 10 bp End Start 7 0 S000 bp 0 5000 bp Min length 100 bp E Start End 0 5000 bp Require init codon true Search in bo ds C End End Genetic code 1 ade The constraints imposed on the results Start Start of the algorithms calculations Alternatively you can create edit a schema using a text editor When the schema has been created and all its parameters have been set you can run it for a nucleotide sequence The resul
321. ting Molecular Surface Selecting Background Color Selecting Detail Level e Enabling Anaglyph View Moving Zooming and Spinning 3D Structure Selecting Sequence Region Selecting Models to Display Structural Alignment Exporting 3D Structure Image Working with Several 3D Structures Views e Chromatogram Viewer e Exporting Chromatogram Data e Viewing Two Chromatograms Simultaneously s ee Graphs Package e Description of Graphs e Graph Settings e Saving Graph Cuttoffs as Annotations e Doitplot e Creating Dotplot Navigating in Dotplot Zooming to Selected Region Selecting Repeat Interpreting Dotplot Identifying Matches Mutations Invertions etc Editing Parameters Filtering Results Saving Dotplot as Image Saving and Loading Dotplot Building Dotplot for Currently Opened Sequence e Comparing Several Doitplots e Alignment Editor e Overview e Alignment Editor Features Alignment Editor Components Navigation Coloring Schemes e Creating Custom Color Scheme Highlighting Alignment Zooming and Fonts Searching for Pattern Consensus e Working e e Export Consensus Alignment Overview with Alignment Undo Redo Framework Selecting Subalignment Moving Subalignment Editing Alignment e Removing Selection Filling Selection with Gaps Replacing with Reverse Complement Replacing with Reverse Replacing with Complement Removing Columns of Gaps e Removing All Gaps Saving Alignment Aligning Sequences Aligning Sequence to this Alignment Pairwise Ali
322. to BWA The dialog looks as follows Build Index Align short reads method Reference sequence Index file name Index algorithm a E Colorspace c There are the following parameters Reference sequence DNA sequence to which short reads would be aligned to This parameter is required Index file name file to save index to This parameter is required Index algorithm a Algorithm for constructing BWA index Available options are It implements three different algorithms e is designed for short reads up to 200bp with low error rate lt 3 It does gapped global alignment w r t reads supports paired end reads and is one of the fastest short read alignment algorithms to date while also visiting suboptimal hits e bwtsw is designed for long reads with more errors It performs heuristic Smith Waterman like alignment to find high scoring local hits Algorithm implemented in BWA SW On low error short queries BWA SW is slower and less accurate than the is algorithm but on long reads it is better e div does not work for long genomes Colorspace color the input is read in colorspace colors are encoded as characters A C G T A blue C green G orange T red BWA SW BWA is a fast light weighted tool that aligns relatively short reads to a reference sequence Click this link to open BWA homepage BWA SW share similar features such as long read support and split alignment BWA SW is embedde
323. to position Select sequence region Ctrl A Ctrl N New annotation Rename item Copy Paste Also you can use the shortcut Ctrl G Toggling Views It is possible to switch the Sequence overview Sequence zoom view and the Sequence details view visibility using the rightmost button in the toolbar The sequence can be removed from the view using the same menu Once you remove the last sequence in the view the view is automatically closed Exporting Sequence Image Use a sequence toolbar Export image button to save a screenshot of the sequence oe e The Export Image dialog will appear where you should set name location export settings and format of the picture 52 Unipro UGENE Manual Version 1 20 0 m Export Image Sequence export settings Area O Currently viewed Zoomed annotations Sequence details Export to file File name UGENE supports export to the BMP JPEG JPG PNG PPM TIF TIFF XBM XPM and SVG image formats You can export currently viewed zoomed annotations or sequence detailes areas Also you can export whole sequence or custom region Use the Region settings to do it Zooming Sequence To zoom a sequence in the Sequence zoom view you can use one of the zoom button on the sequence toolbar There are standard Zoom In and Zoom Out buttons Additionally you can zoom to a selected region using the Zoom to Selection button To restore the default view of the Sequence
324. tools HMM build HMM calibrate and HMM search In the original program the corresponding commands are hmmbuild hmmcalibrate and hmmsearch To access these tools select the Tools HMMER2 tools submenu of the program main menu Tools Window Help Ea Create index File F EL DNA Assembly e ce Weight matrix a F io HMMERS tools d HMM build F 29 SITECON d HMM calibrate n 4a HMMERS tools 001363 sequence HMM search 1 5k WorkFlow Designer We highly recommend reading the original HMMER2 documentation to learn how to use utilities provided by the plugin SSE2 algorithm is implemented by Leonid Konyaev Novosibirsk State University Use of the SSE2 optimized version of the HMM search algorithm with quad core CPU gives gt 30x performance boost when compared with the original single threaded algorithm single sequence mode e Building HMM Model HMM Build e Calibrating HMM Model HMM Calibrate e Searching Sequence Using HMM Profile HMM Search Building HMM Model HMM Build HMM build tool is used to build a new HMM profile from a multiple alignment You can use any alignment file formats supported by UGENE The output HMM profile format is compatible with the HMMER2 package 212 Unipro UGENE Manual Version 1 20 0 T HMM Build Multiple alignment file File to save HMM profile Expert options Name this HMM Default hmmis behaviour Emulate hmmfs beh
325. true allow alternative codons allows ORFs starting with alternative initiation codons accordingly to the current translation table Boolean Optional Default false Example ugene find orfs in human_Tl fa out result gb regquire init codon false Finding Repeats Task Name find repeats Searches for repeats in sequences and saves the regions found as annotations Parameters in semicolon separated list of input files String Required out output file with the annotations String Required name name of the annotated regions String Optional Default repeat_unit min length minimum length of the repeats Number Optional Default 5 identity percent identity between repeats Number Optional Default 100 min distance minimum distance between the repeats Number Optional Default 0 max distance maximum distance between the repeats Number Optional Default 5000 inverted if true searches for the inverted repeats Boolean Optional Default false Example ugene find repeats in murine gb out murine_repeats gb identity 99 Finding Pattern Using Smith Waterman Algorithm Task Name find sw Searches for a pattern in a nucleotide or protein sequence using the Smith Waterman algorithm and saves the regions found as annotations 2 6 Unipro UGENE Manual Version 1 20 0 Parameters in input sequence file String Required out output file with the
326. ts are saved as a set of annotations to the specified file in the Genbank format Also when you have query designer scheme you can analyze a nucleotide sequence from the sequence view with a help of this schema Call the Analyze gt Analyze with query schema context menu item for this To learn more about the Query Designer read the Query Designer Manual Plasmid Auto Annotation Plasmid Auto Annotation feature allows to automatically annotate possible functional elements of the given sequence such as promoters terminators origin of replication known genes common primers and other features Conceptually this functionality is similar to the one offered by PlasMapper software The database for plasmid auto annotation is based on the following resource To activate Plasmid Auto Annotation upon your sequence use the menu item Analyze Annotate plasmid and custom features In the appeare 255 Unipro UGENE Manual Version 1 20 0 d dialog one can selected the features to search in sequence Dy Plasmid Auto Annotations Selected features Promoter Origin Terminator Primer V Gene Other features Regulatory Cerone Gear ee The detected plasmid features are stored as automatic annotations and can be controlled through corresponding menu Refer Automatic Annotations Highlighting to learn more The database containing features and their sequences is located in a subfolder of UGENE da
327. uence ugene genome aligner build index reference path to ref Align short reads using existing index ugene genome aligner reference path to ref short reads path to reads result path to result CLI Predefined Tasks Using current version of UGENE you can perform the following tasks by running a simple command Format Converting Sequences Converting MSA Extracting Sequence Finding ORFs Finding Repeats Finding Pattern Using Smith Waterman Algorithm Adding Phred Quality Scores to Sequence Local BLAST Search Local BLAST Search Remote NCBI BLAST and CDD Requests Annotating Sequence with UQL Schema Building Profile HMM Using HUMER2 Searching HMM Signals Using HMMER2 Aligning with MUSCLE Aligning with ClustalW Aligning with ClustalO Aligning with Kalign Aligning with MAFFT Aligning with T Coffee Building PFM Searching for TFBS with PFM Building PWM Searching for TFBS with Weight Matrices Building Statistical Profile for SITECON Searching for TFBS with SITECON Fetching Sequence from Remote Database Gene by Gene Report Reverse Complement Converting Sequences Variants Calling Generating DNA Sequence Format Converting Sequences Task Name convert seq Converts a sequence from one format to another Parameters in input sequence file String Required out name of the output file String Required format format of the output file String Optional The following values a
328. ull down the latest list of software from ugene archive it knows about including the PPA sudo apt get update e Now you re ready to start installing UGENE sudo apt get install ugene e To install the non free UGENE plugins do the following sudo apt get install ugene non free UGENE will appear in the applications list Unipro UGENE Manual Version 1 20 0 OR UGENE Eig Applications F U Unipro UGENE l Files amp Folders Native Installation on Fedora Ugene packages for different Fedora versions are available on the Fedora To start installing and using software do the following e Open a terminal and enter sudo yum install ugene e Now the latest available UGENE appears in the applications list Basic Functions e UGENE Terminology e UGENE Window Components e Welcome Page e Project View e Task View e Log View e Notifications Main Menu Overview Creating New Project Creating Document Opening Document e Opening for the First Time e Advanced Dialog Options e Opening Document Present in Project e Opening Several Documents Opening Containing Folder Exporting Documents Locked Documents Using Objects and Object Views Exporting Objects e Exporting Sequences to Sequence Format Exporting Sequences as Alignment Exporting Alignment to Sequence Format Exporting Nucleic Alignment to Amino Translation Export Sequences Associated with Annotation Using Bookmarks Exporting Project Search
329. user_password and in the second case execute gt GRANT SELECT ON your_database_name TO user_nickname IDENTIFIED BY user_password 4 Use the database from a UGENE instance The database with your_database_name is now available from a UGENE instance version 1 14 or higher It s time to try it out and fill it with some initial data To do it open UGENE and connect to the database As we need to add the data to the database use user_nickname and user_passwora of a user with privileges to modify the database As soon as connection is established a dd the required data to the database From now on the data will be available for all users from this and other UGENE instances who connected to the same database Connecting to a Shared Database To start using the shared database you need to have a running public MySQL database server Usually the system administrator of your department does it You should ask him or her to give you the access to a MySQL database Particularly you need a few parameters to connect to the database the IP address of the server the computer where a MySQL server is running a user name for the MySQL database and a password You can also install a MySQL server by yourself on any public computer you have an access to even on your workstation following the steps described in the Configuring Database section To connect to the database use the File gt Connect to shared database main menu item The f
330. ut calibration settings provided to UGENE team by the author of S TECON The original TFBS alignments used to calculate profiles can be requested directly from the author of S TECON Types of SITECON Models e Eukaryotic e Prokaryotic Eukaryotic Name CEBP_a CEBP_all CLOCK cMyc_can CRE E2F1 E2F1 DP1sel1 Description CCAAT enhancer binding protein_alpha CCAAT enhancer binding proteins Circadian Locomotor Output Cycles Kaput Myc c Myc is a regulator gene that codes for a transcription factor A mutated version of Myc is found in many cancers Cyclic AMP response element Transcription factor E2F1 is a protein that in humans is encoded by the E2F1 gene E2F factors bind to DNA as homodimers or heterodimers in association with dimerization partner DP1 206 EGR1 EKLf ER2 GATA_all GATA 1 GATA 2 GATA 3 HMG 1 HNF 1 HNF 3 HNF 4 IRF isre MyoD MyOGsel3 NF 1 NF E2 NFATp NFkB_all NFkB_hetero NFkB_ homo Nfy Nrf2 Oct 1 Oct_all p53 PPRF Pu1 setCREB setCREBzag SRE_san SAF STAT1 STAT Unipro UGENE Manual Version 1 20 0 Early growth response protein 1 Erythroid Kruppel like Factor Estrogen receptor beta GATA transcription factors are a family of transcription factors characterized by their ability to bind to the DNA sequence GATA GATA binding factor 1 GATA binding protein 2 Trans acting T cell specific transcription factor GATA 3 High mobility group protei
331. vaialble e Linux e Ubuntu 12 04 or later e Fedora 19 or later e lf you have another Linux system you may use a universal binary package e RAM e 512 Mb RAM is required e At least 2 Go RAM is recommended e Disk space The minimum required disk space depends on the UGENE package e Standard package 200 300 Mb e Full package 500 900 Mb e NGS package 21 24 Gb e Display e It is recommended to set the screen resolution to a value greater than 1280x720 e Internet e Internet connection is required for some tasks like loading data from online databases UGENE takes care to use capabilities of your system the more RAM and cores you have the more quickly you ll get results of your calculations Also if you have an OpenCL capable video card you can use GPU optimized versions of the following tools e Smith Waterman Search e UGENE Genome Aligner UGENE Packages Besides selecting an appropriate package for your operating system Windows Mac OS X or Linux 32 or 64 bit you should take into account the following considerations Should I download standard full or NGS package In most cases the full package is the best choice Exceptions are e Use the standard package if Unipro UGENE Manual Version 1 20 0 e You re going to use only basic UGENE features and don t want to waste Internet traffic e You have limited disk space e Use the NGS package if e You re going to analyze ChIP Seq data with the Cistrome pi
332. vanced options Select search E Search for short nearly exact matches Expectation value 10 000000 gt E Megablast Best hits limit 100 Both strands Direct E Complement Database path Select Base name for BLAST DE files a database file Save annotation s to Existing table gy NC_001363 features murine gb Create new table Use auto annotations table w Annotation parameters Group name lt auto gt Description Number of CPUs being used The following general options are available Select search here you should select the tool you would like to use If the query sequence is a nucleotide sequence then blastn blast x and tblastx items are available For a protein sequence the items are blastp and tblastn Expectation value this option specifies the statistical significance threshold for reporting matches against database sequences Lower expect thresholds are more stringent leading to fewer chance matches being reported Culling limit the maximum number of hits that will be shown not equal to number of annotations The maximum availablle number is 5000 Search for short nearly exact matches automatically adjusts the word size and other parameters to improve results for short queries Megablast select this option to compare query with closely related sequences It works best if the target percent identity is 95 or more but it is very fast Database path path to the database files Base name fo
333. verhang Preview Fragment of human T1 fa 5 i ITTTASAAT CEG AADATTTAGCE Here you can select the type of each DNA end and even input a custom overhang The changes you ve made are shown in the Preview area of the dialog To confirm the changes and close the dialog click the OK button Reverse Complement a Fragment To reverse complement a fragment check the nverted check box for the fragment in the new molecule contents list Other Constuction Options To save the fragments of the new molecule as annotations check the Annotate fragments in new molecule check box To make all DNA ends blunt check the Force blunt and omit all overhangs check box All overhangs would be cut in this case Check the Make circular check box to make the new molecule circular Output 196 Unipro UGENE Manual Version 1 20 0 On the Output tab of the dialog you can select the file to save the new molecule to The molecule is opened by default as soon as it is created To modify this behavior uncheck the Open view for new molecule check box on the same tab To save the molecule file to the hard disk immediately after it is created check the Save immediately check box Otherwise it would be stored in memory until you save or remove it Creating PCR Product To create a PCR product from a primer use the Cloning gt Create PCR product context menu of primer annotation The Create PCR Product dialog appears Create PCR product Frag
334. verse complement select it and use the Edit gt Replace with reverse complement item in the context menu Replacing with Reverse To replace sequence s in the alignment with reverse select it and use the Edit gt Replace with reverse item in the context menu Replacing with Complement To replace sequence s in the alignment with complement select it and use the Edit gt Replace with complement item in the context menu Removing Columns of Gaps To remove colums containg certain number of gaps select the Edit Remove columns of gaps item in the context menu The dialog appears tis Remove Columns of Gaps 0 Remove columns with number of gaps 1 Remove columns with percentage of gaps 10 Remove all gap only columns There are the following options Remove columns with number of gaps removes columns with number of gaps greater than or equal to the specified value Remove columns with percentage of gaps removes columns with percentage of gaps greater than or equal to the specified value Remove all columns of gaps this option is selected by default It specifies to remove columns from the alignment if they entirely consist of gaps Select the option required and press the Remove button Removing All Gaps Use the Edit Remove all gaps item in the Actions main menu or in the context menu to remove all gaps from the alignment Saving Alignment To save current alignment click the Save alignment button to the the alignme
335. w could be used as a profile 220 Unipro UGENE Manual Version 1 20 0 Consensus ATGGCACATCCCtCaCAt cTAGGaTTCcCAAGAcGC CcTCact 1 2 4 6 6 10 12 14 16 18 820 22 24 26 26 30 32 3 J 35 40 43 Loach y i i 1 A i i Lf 3 i Tuna Trout Eel The same profile after profile to profile alignment Consensus ATGGCacatcCCctcaCAac tAGGttTt t cCAAGAcGCagtcTtCacCca 12 2 6 Loach 1 Tuna 1 Trout 1 Eel 1 Seahorse 1 Salamander 1 Frog 1 Panda 1 There are two gap columns inserted into the source profile and two gap columns inserted into the added one Therefore the profiles columns kept intact and the alignments haven t been changed Aligning a profile to the active alignment you will modify the original alignment file since it will contain 2 profiles after the operation is completed Aligning Sequences to Profile with MUSCLE Another feature provided by the plugin is aligning a set of unaligned sequences to an existing profile To use this feature select the Align Align sequences to profile with MUSCLE context menu item This option is not available in the original MUSCLE package v3 7 and is a new functionality for original MUSCLE users In this mode each sequence from the input file is aligned to the active profile separately and is merged to the result alignment only after all sequences are processed For example the alignment in the picture above can be used as a profile again And the added pr
336. w only one model check the item and click the OK button To show several models select it and click OK button To show the inverted selection click the nvert button and click OK button Structural Alignment To use the structural alignment call the Structural alignment gt Align with context menu item The following dialog will appear Algorithm Poos y Reference Mobile Structure 1CF7 Structure Chain ins Chain All chains z Redi Model Here you can change reference and mobile settings After that click on the OK button To reset structural alignment call the Structural alignment gt Reset context menu item 96 Unipro UGENE Manual Version 1 20 0 Exporting 3D Structure Image To export a 3D structure image select the Export Image item in the 3D Structure Viewer context menu or in the Display menu on the toolbar The Export Image dialog will appear 8 Export Image File name C Users user untitled jpal Format jpg Width 1854px Height 343px F Quality Here you can browse for the file name select the width and height of the image as well as its format svg png ps jpg jpeg tiff tif pdf bmp or ppm For jpg jpeg formats the quality score parameter is available Working with Several 3D Structures Views To add another view to the 3D Structure Viewer you can e Drag a required 3d object from the Project View to the 3D Structure Viewer Project x S 3D Structure Viewer Active view Object
337. with a help of the corresponding buttons in the right corner of the dialog e RTPCR Primer Design RTPCR Primer Design This feature allows to search for primer pairs that span introns on the genomic sequence or exon junctions on the mRNA sequence Note that RT PCR design is only available for mRNA cDNA sequences with annotated exons There are several ways to obtain the cDNA for a corresponding DNA sequence e From NCBI or ENSEMBL database For example one can download the TMPRSS2 transcript variant 1 from NCBI Genbank using identifier NM_001135099 1 This can be also done from UGENE using option Access remote database or Search NCBI Genbank e Align the genomic and cDNA sequences using spliced aligner For this option one must have both genomic and cDNA sequences In UGENE the spliced alignment can be performed using the Spidey tool To run the alignment open the genomic sequence and select action Align Align to mRNA sequence The generated exon annotations can be then exported using action Export Export sequence of selected annotations To design primers for your mRNA sequence and go to the RT PCR tab of the Primer Designer dilaog 251 Unipro UGENE Manual Version 1 20 0 Primer Designer Main General Settings Internal Oligo Penalty Weights RT PCR Sequence Quality Result Settings 4 Design primers for RT PCR analysis mRNA sequence Exon annotation name exon V Minimum exon junction overl
338. y and result files and click on the Convert button CAP3 CAP3 CONTIG ASSEMBLY PROGRAM Version 3 is a sequence assembly program for small scale assembly with or without quality values Click this link to open CAP3 homepage CAP3 is embedded as an external tool into UGENE Open Tools DNA assembly submenu of the main menu cece Window Help r Align short reads binm 32 Build index P i T Convert UGENE Assembly data base to SAM format Multiple alignment j H ii t DNA assembly Contig assembly with CAP3 Select the Contig assembly with CAP3 item to use the CAP3 The Contig Assembly With CAP3 dialog appears E Contig Assembly with CAP3 Input files long DNA reads to assembly 241 Unipro UGENE Manual Version 1 20 0 You can add or remove input files using Add and Remove buttons To remove all files click the Remove all button Input files are files with a long DNA reads in FASTA FASTQ SCF or ABI formats At least one input file should be added Input a Result contig name and press the A un button CAP3 produces assembly results in the ACE file format ace The file contains one or several contigs assembled from the input reads The quality scores for FASTA sequences can be provided in an additional file The file must be located in the same folder as the original sequences and have the same name as FASTA file but another extension qual Also you can change the following
339. ylogenetic Tree dialog for the MrBayes method has the following view 137 Unipro UGENE Manual Version 1 20 0 Build Phylogenetic Tree Chain length 10000 Subsampling frequence 1000 Burn in length 10 Heated chains 4 Heated chain temp Random seed save tree to Remember Settings Restore Default There are two steps to a phylogenetic analysis using MrBayes 1 Set the evolutionary model 2 Run the Markov chain Monte Carlo MCMC analisys The evolutionary model is defined by the following parameters Substitution model specifies the general structure of a DNA substitution model This parameter is available for the nucleotide sequences It corresponds to the Nst setting of MrBayes You may select one of the following JC69 Nst 1 HKY85 Nst 2 GTR Nst 6 Rate matrix fixed specifies the fixed rate amino acid model This parameter is available for amino acid sequences The following models are available e poisson e jones e dayhoff e mtrev e mtmam e wag e rtrev e cprev e vi e blosum e equaline The following parameters are common for nucleotide and amino acid sequences Rate sets the model for among site rate variation Select one of the following e equal no rate variation across sites e gamma gamma distributed rates across sites The rate at a site is drawn from a gamma distribution The gamma distribution has a single parameter that describes how much rates vary
340. ze ions as tations Result strand Direct Complement Both E Annotation must fit into region Found 2 regions Using this dialog you can search for DNA sequence regions that contain every annotation from the list on the left side The found regions are displayed on the right side of the dialog Use the Save regions as annotations button to store the regions as new annotations to the sequence DNA Flexibility To search for regions of high DNA helix flexibility in a DNA sequence open the sequence in the Sequence View and select the Analyze Find high DNA flexibility regions item in the context menu Note that only standard DNA alphabet is supported i e the sequence should consist of characters A C G T and N The following dialog appears 167 Unipro UGENE Manual Version 1 20 0 U DNA Flexibility Search Settings High DNA Flexibility Regions Settings Window size Window step Threshold The calculation is made for overlapping windows along a given sequence If there are two or more consecutive windows with an average flexibility threshold in each window greater than the specified Threshold parameter such area is marked by an annotation The average threshold in a window is calculated by the following formula average window threshold sum of flexibility angles in the window the window size 1 The following flexibility angles are used during the calculation Dinucleotide Angle Dinu
341. zoa Ensembl Plants Ensembl Protists CHILE Mouse Genome Informatics VEGA Click on the map to view the list of databases DATABASE SEARCH Search by type Search by organism Search by database name A Z Genome Gene annotation gt Protein sequence and structure gt Interaction and pathways gt Gene expression Notice that an example Ensembl ID below the search bar is highlighted it has a light blue background Current version of the UGENE extension allows detecting the following types of identification numbers 1 Ensemble Gene ID 2 Ensembl Protein ID 3 PDB ID 297 Unipro UGENE Manual Version 1 20 0 Right click on the ID and select Open in UGENE item in the context menu BioMart Central Portal Home IDENTIFIER SEARCH ad Examples KRAS ENSGOGOoo tee Open link in new tab Open link in new window Open link in incognito window Gene retrieval Save link as Copy link address Cancer genes Ensembl Ensembl Bacteria Ensembl Fungi Ensembl Metazoa Ensembl Plants Ensembl Protists Mouse Genome Informatics VEGA i Open in UGENE Inspect element The sequence with the selected ID will be opened in UGENE Opening selected data in UGENE Imagine that you have browsed for required data e g a sequence with annotations and opened for example an html view for the data in a web browser Now you would like to open the data in UGENE to analyze them in more detail Or altern

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