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Geneious User Manual

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1. CCCATCAACAA le 4 Bob TTCTTTCATGGG CAAGA TTT GGGTACC ACCCAAGTATTGACT CCACCCATCAACAA Ce 5 Jane TTCTTTCATGGGGAACAGGCAGATTTT GGGTACC ACCCAAGTATTGACT CACCCAT CAACAA 70 80 90 100 110 120 130 i CCGCTATGTATT TCGTACATTA CTGCCAGCCACCATGAATATTG NNNTA Identity Te 1 Adam GATCCGCTATGTATT TCGTACATTA CTGCCAGCCACCATGAATATT GAGCAAGTACATA Ce 2 Harry CCGCTATGTATT TCGTACATTAAACTT CTGCCAGCCACCATGAATATTG TTGTA Ce 3 Sally CCGCTATGTATT TCGTACATTAAACTT CTGCCAGCCACCATGATGATTG TTGTA le 4 Bob CCGCTATGTATT TCGTACATTA CTGCCAGCCACCATGAATATTG TA Ce S Jane CCGCTATGTATTTCGTCGTACATTA CTGCCAGCCACCATGAATATTG TA 140 150 160 170 180 190 200 CGGTACCATAAATA CTN TGACCACCT GTAGTACAT AAAAACCCAAT CCACAT CAAAACCC erty A gt Pa 1 Adam CCCTACCATCADTA CT mmm PECROCACOTECCTACTACATADADACECACCOAATOCACATACADAACOO Alt click on a sequence position or annotation or select a region to zoom in Alt shift click to zoom out Figure 9 6 The identity graph for an alignment of nucleotide sequences Trimmed regions in sequences are not included when deciding when to strip a column 9 4 Consensus sequences To display a consensus sequence on your alignment check the Consensus option under the Display tab The consensus sequence is displayed above the alignment or assembly and shows which residues are conserved are always the same and which
2. 10 CHAPTER 1 GETTING STARTED We recommend at least the following specifications for running Geneious note that these are minimum requirements for working with large datasets such as NGS sequences you will need a higher spec machine e Processor Intel x86 x86_64 e Memory 2048MB or more e Hard disk 2GB or more free space e Video 1024x768 resolution or higher Geneious also needs Java 1 6 or higher to run Geneious comes bundled with the correct version of Java for your OS If you require a version of Geneious that does not include Java please contact Geneious Support Once Geneious has downloaded double click on the Geneious icon to start installing the pro gram While this is happening you will be prompted for a location to install Geneious Please check that you are satisfied with the location before continuing If you are using Mac OS X you will only have to double click on the disk image that is down loaded then drag the Geneious application to your Applications folder Don t run Geneious from the mounted disk image as there are no write permissions on this You must drag the icon into your Applications folder and run it from there 1 2 Geneious setup 1 2 1 User preferences User preferences can be changed by going to Tools Preferences This window can also be opened using the shortcut keys Ctrl Shift P Windows Linux or command Shift P Mac OS X In the user preferences you can change data storage memory an
3. 114 CHAPTER 9 SEQUENCE ALIGNMENTS 9 2 2 Multiple sequence alignments A multiple sequence alignment is a comparison of multiple related DNA or amino acid se quences A multiple sequence alignment can be used for many purposes including inferring the presence of ancestral relationships between the sequences It should be noted that protein sequences that are structurally very similar can be evolutionarily distant This is referred to as distant homology While handling protein sequences it is important to be able to tell what a multiple sequence alignment means both structurally and evolutionarily It is not always possible to clearly identify structurally or evolutionarily homologous positions and create a single correct multiple sequence alignment Durbin et al 1998 Multiple sequence alignments can be done by hand but this requires expert knowledge of molecular sequence evolution and experience in the field Hence the need for automatic mul tiple sequence alignments based on objective criteria One way to score such an alignment would be to use a probabilistic model of sequence evolution and select the alignment that is most probable given the model of evolution While this is an attractive option there are no efficient algorithms for doing this currently available However a number of useful heuristic algorithms for multiple sequence alignment do exist Progressive pairwise alignment methods The most popular and time efficient
4. EcoBl per match per match EcoBl per match CpG CpG Dcm Dcm Dam Dcm Dam EcoKI per match per match CpG per match per match Dem C 1 per match CpG per match EcoBl per match CpG Ec 1 per match per match free database Save Selected Enzymes cancel Goes 185 186 CHAPTER 14 CLONING e If you have selected only one nucleotide sequence document and it has annotated re striction sites you can select Annotated cut positions to cut the document on these sites When this option is selected the options to filter the enzymes by their effective recog nition sequence length or number of hits are disabled You can select a subset of the annotated enzyme sites under More Options e If you do not have annotated cut sites already on your document you can choose Enzyme set and select which enzyme s you wish to use This runs Find Restriction Sites first but does not generate restriction site annotations on your original sequence See the section on Find Restriction Sites 14 1 for more detail e Where multiple enzymes are selected you can either digest the original sequence by Each enzyme separately which returns a separate sequence list of the fragments produced for each enzyme or by All enzymes at once which digests by all selected enzymes in one operation 14 3 Restriction Cloning To do basic restriction cloning inserting a digested fragment into a vector select Restriction Cloning
5. Enzymes annotated on insert This option lets you use only the enzymes used to cut the insert fragment Enzyme set This option lets you use the enzymes from a predefined enzyme set eg the enzyme set you have created containing the enzymes you have in your lab e Cut vector with Whenever you change the options for the polylinker or candidate en zymes Geneious will recalculate the compatible enzymes on the vector It will look for enzymes which meet one of the following conditions in addition to cutting only within the polylinker and belonging to enzymes from the candidate enzyme set 1 A single enzyme which cuts the vector once such that the insert can be inserted in the gap Possible only when the insert has complementary cut sites 2 A single enzyme which cuts the vector twice such that the insert can be inserted into the gap vacated by the fragment between the two cut sites 3 Two enzymes which each cut the vector once such that the insert can be inserted into the gap vacated by the fragment between the two cut sites Other Options The Product section of the options displays a diagram showing the ligation points in the inser tion The parts of the ligation points belonging to the vector appear in bold in this diagram Below this is a checkbox where you can choose whether to Keep fragments which are not part of the product If this box is checked a document will be created representing the fragment removed from the vector
6. Must cut between may cut outside Returns enzymes which cut between the spec ified bases and may also cut outside that region Must not cut between Returns enzymes which only cut outside the specified bases e Advanced This displays a table of all enzymes in your candidate set including their recognition site overhang and methylation information Figure 14 1 Only the enzymes selected in this table will be considered in the analysis initially all rows are selected You can click on the column headers to sort the table ascending or descending by that column and you can Shift click and Ctrl click to select a range of rows and to toggle the selection of a row respectively e To create custom enzyme set select the enzymes you want in the Advanced window then click Save Selected Enzymes and give the set a name This set will then be available in the Candidate Enzymes lists After configuring your options click Apply to record the restriction enzyme site annotations on the sequence The annotation shows the enzyme s recognition site and the cut site Once the document is saved two new tabs will appear above the sequence view Enzymes displays the list of enzymes and their cut positions Fragments displays a list of fragments that would be produced from the restriction digests These tables can be exported as csv files for subsequent processing with other software such as e g Microsoft Excel To select the region between two
7. These can be downloaded from the plugins page on our website or within Geneious by going to Plugins under the Tools menu For more information on running these programs please consult the user manual for the source software 12 4 Resampling Bootstrapping and jackknifing Resampling is a statistical technique where a procedure such as phylogenetic tree building is repeated on a series of data sets generated by sampling from one original data set The results of analyzing the sampled data sets are then combined to generate summary information about the original data set In the context of tree building resampling involves generating a series of sequence alignments by sampling columns from the original sequence alignment Each of these alignments known as pseudoreplicates is then used to build an individual phylogenetic tree A consensus tree can then be constructed by combining information from the set of generated trees or the topologies that occur can be sorted by their frequency see below Bootstrapping is the statistical method of resampling with replacement To apply bootstrap ping in the context of tree building each pseudo replicate is constructed by randomly sampling columns of the original alignment with replacement until an alignment of the same size is ob tained see Felsenstein 1985 Jackknifing is a statistical method of numerical resampling based on deleting a portion of the original observations for each pseudo replic
8. To see why no primers or DNA probes were found for particular sequences click the Details button at the bottom of the dialog The dialog will then open out to display a list of all the sequences for which no primers or DNA probes were found For each of the sequences the following information is listed e Which of Forward Primer Reverse Primer Primer Pair and or DNA Probe could not be found in the sequence e For each of these specific reasons for rejection are listed eg Tm too high or Unaccept able product size along with a percentage which expresses how many of the candidate primers or probes were rejected for this reason After examining the details you can choose take no action or continue and annotate the primer and or DNA probes on the sequences which were successfully designed for 172 CHAPTER 13 PCR PRIMERS 13 2 Manual primer design It is possible to create PCR primers by adding a primer annotation directly onto a sequence This is especially useful for cloning applications as generally the primers must bind to a speci fied set of bases at the beginning and end of the gene to be cloned To manually add a primer select the region of sequence where you wish the primer to bind and click Add Annotation Make the annotation type primer bind and you will then see primer specific options and char acteristics as in Figure 13 4 Changing the primer binding site position in the Add annotation window will automatic
9. Topology Threshold The percentage of topologies in the original trees which must be represented by the summarizing topologies e Save raw trees If this is turned on then all of the trees created during resampling will be save in the resulting tree document The number of raw trees saved will therefore be equal to the number of samples 12 3 Tree building methods and models 12 3 1 Neighbor joining In this method neighbors are defined as a pair of leaves with one node connecting them The principle of this method is to find pairs of leaves that minimize the total branch length at each stage of clustering starting with a star like tree The branch lengths and an unrooted tree topology can quickly be obtained by using this method without assuming a molecular clock see Saitou amp Nei 1987 12 3 2 UPGMA This clustering method is based on the assumption of a molecular clock It is appropriate only for a quick and dirty analysis when a rooted tree is needed and the rate of evolution is does not vary much across the branches of the tree see Mitchener amp Sokal 1957 12 3 3 Distance models or molecular evolution models for DNA sequences The evolutionary distance between two DNA sequences can be determined under the assump tion of a particular model of nucleotide substitution The parameters of the substitution model 12 3 TREE BUILDING METHODS AND MODELS 157 define a rate matrix that can be used to calculate the probability of e
10. Alternatively an experimental plugin for merging paired reads using FLASH is available from the Geneious website plugins page 10 1 6 Splitting multiplex barcode data Multiplex or barcode data e g 454 MID data can be separated using Separate Reads by Barcode from the Sequence menu This function copies all sequences matching a given barcode to a correspondingly named sequence list document Default settings are provided for 454 standard and Titanium MID barcodes with or without Adaptor B trimming and Rapid MID barcodes These settings recognise standard MID se quences provided by 454 and use their names when appropriate To enter a custom barcode set select Custom settings then under Barcode set choose Edit barcode sets Then click Add to add your list of barcodes To specify fixed sequences either side of the barcode enter these in the Adaptor and Linker sections If you only want to extract sequences with a single specific barcode sequence e g a primer 10 1 READ PROCESSING 131 Use default settings for l Custom E Adapter Fixed Length 4 BP e Sequence TCAG Barcode M Allow single mismatch in barcode or adapter Barcode Set My barcodes Minimum Reads Per Barcode 8 Specific Barcode O Length 10 BP rae Automatic Minimum Reads Per Barcode _ _ Percentage of reads e First sequence in mate pair Barcode is on f _ Second sequence in ma
11. These details can also be found in your browser s settings 5 Set the proxy host and port settings under the General tab to match those in your browser 6 If your proxy server requires a username and password you can specify these by clicking the Proxy Password button directly below Note If you are using any other browser and cannot find the proxy settings please use the Support Button in the Geneious toolbar to contact Geneious support Connection settings Use browser connection settings B Proxy host Proxy port Config file location Proxy Password 4 Proxy Help Figure 1 2 Proxy settings in Preferences 1 2 6 Installing plugins and customizing features You can extend the functionality of Geneious with a variety of plugins These can be down loaded from our website or managed via the plugins and features preferences Figure 1 4 in Geneious To access the plugins preferences go to Tools Plugins This window contains a list of available plugins in the top window which lists plugins avail able for download which aren t already installed To install a plugin click the Install button and for more information about the plugin click Info Plugins with a star are note worthy plugins as chosen by the Geneious team 16 CHAPTER 1 GETTING STARTED e 4 Tools Preferences Preferences Settings Internet Advanced Advanced Advanced Options Proxies Network Network Connect
12. of the se lected sequence On a linear sequence this is used to indicate that a sequence is a subse quence of some larger sequence On a circular sequence this is used to shift the origin of the sequence Convert between DNA and RNA Changes all T s in a sequence to U s or vice versa depending on the type of the selected sequence Once this is performed click Save in the Sequence View to make the change permanent Set Paired Reads Sets up paired reads for assembly See section 10 1 4 Merge Paired Reads Merges paired reads using BBMerge see section 10 1 5 Remove Duplicate Reads Uses Dedupe to remove duplicate sequences from NGS datasets see section 10 1 2 Error Correct and Normalize Uses BBNorm to error correct and normalize NGS reads see section 10 1 3 Set Read Direction Marks sequences as forward or reverse reads so the correct reads are reverse complemented by assembly Separate Reads by Barcode separates multiplex or barcode data e g 454 MID data See section 10 1 6 2 6 GENEIOUS MENU BAR OPTIONS 31 e Group Sequences into a List creates a sequence list containing copies of all of the selected sequences See section 5 1 1 e Extract Sequence from List copies each sequence out of a sequence list into a separate sequence document 2 6 6 Annotate amp Predict Menu This menu contains many tools for finding predicting and annotating regions of interest in sequences and alignments Plugins that involve
13. The protein translation can be viewed alongside the nucleotide sequence by checking the Trans lation option in the m Display tab Select the genetic code and reading frame s you require You can also choose to translate relative to selection or annotations such as CDS Figure 5 5 In an alignment the sequence frame can be calculated relative to the individual sequences the alignment the consensus or a specific reference sequence On a contig or alignment the trans lation can be displayed on the consensus and reference sequence only or it can be displayed on all sequences If you wish to view only the translation and turn off the nucleotide sequence uncheck Nu cleotides However this is only for display purposes if you wish to work with the translation in downstream analysis you must extract it to a separate document using the Translate button above the sequence viewer The Translate button will create new protein document from the translated DNA using your choice of reading frame and genetic code This option can also be accessed from the Sequence menu 78 CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES Y Hide Sequence View Annotations Dotplot Self DNA Fold Text View Info lt a dy E Extract GRC Translate Add Edit Annotation g Allow Editing Annotate amp Predict HM Save eg 1 q A alle 89 e A 22 Display 10 20 181 190 200 210 2 E coli K12 MG1655 ACAACATCCAT GAAACGCAT TAGCACCAC
14. or millions of reads generated by next generation sequenc ing machines To assemble a contig firstly select all of the sequences and or contigs you wish to assemble in the document table then click Align Assemble in the toolbar and choose De Novo Assemble The basic options for de novo assembly will then be displayed The options available here are as follows e Assemble by aka Assemble by Name If you have selected several groups of fragments which are to be assembled separately you can specify a delimiter and an index at which the identifier can be found in all of the names Sequences are grouped according to the identifier and each group is assembled separately If a reference sequence is specified it is used for all groups eg For the names A03 1 ab1 A03 2 ab1 B05 1 ab1 B05 2 ab1 etc where A03 and B05 are the identifiers you would choose Assemble by 1st part of name separated by full stop e Use of data This option is will show with large datasets and enables you to assemble a subset of your data rather than the full dataset For example if you enter 20 here then the first 20 of reads in a sequence list will be assembled and the rest will be ignored This is useful in situations where the full dataset is too large for the size of genome being assembled e Assembly method In this section you can choose from the built in Geneious assembler or Tadpole Velvet MIRA and CAP3 assemblers if you have these plu
15. translations section 5 5 and analysis graphs section 5 2 7 are also displayed in this viewer 5 2 THE SEQUENCE VIEWER 63 Annotations Dotplot Self DNA Fold Text View _ Info lt a ely Er Extract GRC BP Translate Add Edit Annotation VF Allow Editing Annotate amp Predict El Save a Dio 1 50 100 150 200 250 300 350 Tr T a PIPA 14 5 P ot source Mus musculus a Na a Na Na M Show Annotations 5 rAd Q v E 400 450 500 550 600 650 700 X ES ld e ll e le rr leo rro mod Y CDS 1 poke all M Exon 2 o y lt gt ry M mRNA 1 mu gt Source 1 mu y lt gt amp e FR Columns fal Track c Pop out Name Type w exon 4 exon e 1 Cys peroxiredoxin CDS 1 Cys peroxiredoxin mRNA exon 3 exon kas 1 150 5 300 1 35 4 source Mus musculus source A Length 1 596 Freq 1 A 413 25 9 C 342 21 4 G 370 23 2 T 471 29 5 es Alt click on a sequence position or annotation or select a region to zoom in Alt shift click to zoom out Figure 5 2 A view of an annotated nucleotide sequence in Geneious 5 2 1 Zoom level Controls for zooming in and out on sequences are located at the top of the side panel to the right of the sequence viewer The plus and minus buttons increase and decrease the magnifi cation of the sequence by 50 or by 30 if the magnification is already above 50 To zoom in or out by a smaller amount hold down the a
16. Chimpanzee Human Monkey Baboon Mangabey Starling Catbird 10 Sparrow 11 Warbler 12 Gnateater 13 Ostrich Moa 15 Chicken 16 Spoonbill 17 Ibis 18 Lemur 19 Panda 20 Armadillo Polar bear F 00D CCAAGACGCINC CCAAGACG CEC AA GASK CINGIC y ITCAA CAMS CAA CM CCCCMAT CA IERACAN CAAGACGCAACAT CHIC CHAT CAT AGAAGAA MICAAGACGCAACHL See eee eee RRE RRE RRE RRE EEEE de Seal ATCGCATACCCOOMIT AET ACC IE ECA CAMCCIA CI CCA TIO TACAACAR Alt click on a sequence position or annotation or select a region to zoom in Alt shift click to zoom out Figure 2 1 The main window in Geneious 21 Q A Search Help Tutorial tito Alignment View Help The sequence view is a highly customizable viewer for protein and nucleotide sequences sequence alignments circular sequences and phylogenetic trees Zooming The sequence view lets you zoom in to view ini zoom out to vie ce and all its ann br controlling ed at the top of pn the right of thi ou can also hold Alt or Ctrl and turn the mouse wheel up down to zoom in out or Alt click to Zoom in or Alt Shift click to zoom out Selecting and Editing Selection and editing in the sequence viewer is very similar to standard text editing and word processing programs Click and drag to select a region You can drag up and down to select and edit across multiple sequences in an alignment Clicking
17. acidic D E Blue Polar basic K R H 5 2 7 Graphs The alll Graphs tab enables you to display a range of additional metrics on your sequences The type of graphs which are available depend on the type of sequence you are viewing and are listed in the sections below For information on alignment graphs see section 9 3 2 The number control to the right of each graph controls the height of that graph in pixels Sliding window size Many types of graph use a sliding window when calculating values This calculates the value of the graph at each position by averaging across a number of surrounding positions When the value is 1 no averaging is performed When the value is 3 the value of the graph is the average of the residue value at that position and the values on either side CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES Amino acid Hydrophobicity Phe F 1 000 Red Leu L 0 943 lle I 0 943 Tyr Y 0 880 Trp W 0 878 Val V 0 825 Met M 0 738 Pro P 0 711 Cys C 0 680 Ala A 0 616 Gly G 0 501 Purple Thr T 0 450 Ser S 0 359 Lys K 0 283 Gln Q 0 251 Asn N 0 236 His H 0 165 Glu E 0 043 Asp D 0 028 y Arg R 0 000 Blue Figure 5 4 Hydrophobicity values for amino acids and corresponding color scale 5 2 THE SEQUENCE VIEWER 71 Nucleotide sequence graphs GC content This plots a graph of the GC content of the sequence within a window of specified length as the window is moved along the sequence If Frame Plo
18. annotations unique to set A B A annotations unique to set B M A amp B annotations common to both sets Use properties from Both sets prefix properties that differ Example sta 50 WF GT See CD CB D A B cm o _B B A aa C B aD a 1 196 775 19 3 196 766 20 3 196 756 30 3 196 746 Escherichia coli variants y Polymorphism amp CDS 40 3 196 736 CGGAGCCTGAACCTACG CCGGAACCGACACCTGATCCTGAGCCAACACC 107 Figure 8 6 Example 1 Compare annotations setup above and results below for finding polymorphisms within a CDS 108 CHAPTER 8 WORKING WITH ANNOTATIONS Annotation Types Set A Annotation type Polymorphism on track dad Set B Annotation type Polymorphism on track mum 3 M Set C Annotation type Polymorphism on track son Comparison Y Names must match _ All properties must match Y Allow intervals to partially match and produce Partial Annotations Results C A B C annotations unique to set A C B A C annotations unique to set B M C A B annotations unique to set C A amp B amp C annotations common to all sets Use properties from set A Example Set A Set B Set C A B C B A C 50 1 10 20 30 40 3 196 775 3 196 766 3 196 756 3 196 746 3 196 736 3 196 7 Escherichia coli CCGGAGCCTGAACCT
19. bar is blue and the Highlight below bar is yellow Regions where the alignment or assembly is made up of sequences in a single direction e g forward or reverse sequences only can be highlighted by checking Highlight single strand The scale bar to the left of the graph shows minimum and maximum coverage for the entire alignment or assembly as well as a tick somewhere in between for the mean coverage Sequence Logo This displays a sequence logo where the height of the logo at each site is equal to the total information at that site and the height of each symbol in the logo is proportional to its contribution to the information content When zoomed out far enough such that the hori zontal width of each site is less than one pixel then the height is the average of the information 9 3 ALIGNMENT VIEWING AND EDITING 121 200 00 400 000 500 000 800 000 1 001 027 Consensus AR 107 Coverage 0 NC_000913_1st_million nn SNE Consensus 0 NC_000913_1st_million dr Figure 9 5 The coverage graph for an assembly shown zoomed out in the top panel and zoomed in below over multiple sites When gaps occur at at some sites the height is scaled down further to be proportional in height to the number of non gap residues Identity This displays the identity across all sequences for every position Green means that the residue at the position is the same across all sequences Yellow is for less than complete identity and red refers
20. binary files outside of Geneious You can download them from here ftp ftp ncbi nlm nih gov blast executables blast LATEST Choose the appropriate file for your operating system download and extract it You will need to let Geneious know where to look for the files once you have done this To do this go to Tools Add Remove Databases Set Up BLAST Services and select Custom BLAST from the Service drop down box Enter your data location or click Browse to browse to the location of the files 15 4 3 Adding Databases Now that you have set up the executables it is time to add databases to your BLAST Creating a database from a fasta file To create a database from the sequences in a FASTA file go to Tools Add Remove Data bases Add BLAST Database and select Custom BLAST from the Service drop down box Figure 15 5 Choose to Create from file on disk and then click Browse to navigate to the FASTA file that contains the sequences you want to BLAST Enter a name for the database and click OK There are two requirements for a FASTA file to be suitable for creating a database from e The FASTA file must contain only the same types of sequence i e Nucleotide or Amino Acid e The sequences in the FASTA file must all have unique names 208 CHAPTER 15 BLAST Service Custom BLAST Custom BLAST is not set up at the location below Database Location Users helen Geneious 7 0 Data BLAST Let Geneious do
21. but mixing a gap with any other residue in the consensus always produces the total ambiguity symbol N and X for nucleotides and amino acids respectively When the aligned sequences contain quality information in the form of chromatograms or fastq data you can select Highest Quality to calculate a majority consensus that takes the relative residue quality into account This sums the total quality for each potential base call and if the total for a base exceeds 60 of the total quality for all bases then that base is called You can also choose to map the quality of the sequences onto the consensus Choose Highest to map the quality of the highest quality base at each column onto the consensus Select Total to map the sum of the contributing bases minus the sum of the non contributing bases For example if there are two G s and three A s in a column with the G s having qualities of 16 and 24 and the A s having qualities of 40 42 and 50 respectively then because 40 42 50 gt 60 of 40 42 50 16 24 then an A will be called for the consensus This consensus A will have a quality of 40 42 50 16 24 92 if using Total or 50 if using Highest A more complicated example for Highest Quality consensus calling using Total Assume a col umn contains 2 A s with qualities of 30 and 25 1 G with quality 30 and 1 T with quality 15 Because the total qualities of the A s is 55 out of 100 for the column this is not h
22. e Rough Tm A rough calculation of the melting point for a nucleotide sequence using the following calculations If the sequence is less than 14bp in length RoughT m 4 x GCcount 2 x AT count If the sequence is greater than 13bp in length RoughTm 64 9 41 x GC count 16 4 length e Molecular Weight For protein sequences the following values are used for the amino acids A 71 0788 R 156 1875 N 114 1038 D 115 0886 C 103 1388 E 129 1155 Q 128 1307 G 57 0519 H 137 1411 I 113 1594 L 113 1594 K 128 1741 M 131 1926 F 147 1766 P 97 1167 S 87 0782 T 101 1051 W 186 2132 Y 163 1760 V 99 1326 U 150 0388 O 237 3018 For DNA sequences the following values are used A 313 21 T 304 2 G 329 21 C 289 18 The DNA molecular weight assumes no modification of the terminal groups of the se quence If the sequence is a single stranded synthesised oligonucleotide e g by primer exten sion the value is adjusted for the removed phosphate group by using Molecular Weight calculated molecular weight 61 96 If the sequence is a single stranded sequence cut by a restriction enzyme the value is adjusted for the extra 5 monophosphate left by most restriction enzymes by using Molecular Weight calculated molecular weight 61 96 79 0 For dsDNA these values are adjusted for both strands For RNA sequences the following values are used A 329 21 U 306 2 G 345 21 C 305 18 The RNA molecular weight assumes no modification of the terminal g
23. e The contig is assumed to have been fine tuned around indels e Ambiguity characters are ignored other characters in the column are still used e Homopolymer region qualities are reduced to be symmetrical across the homopolymer For example if a series of 6 G s have quality values 37 31 23 15 7 2 then these are treated as though they are 2 7 15 15 7 2 This is done because variations may be called at either end of the homopolymer and because reads may be from different strands e Gaps are assumed to have a quality equal to the minimum quality on either side of them after adjusting for homopolymers e When finding variations relative to a reference sequence the p value calculated is for the variant not the change In other words the p values calculated are independent of the reference sequence data e The approximate p value method calculates the p value by first averaging the qualities of each base equal to the proposed SNP and averaging the qualities of each base not equal to the proposed SNP e Example Assume you have a column where the reference sequence is an A and there are 3 reads covering that position 1 read contains an A in the column and the other 2 reads contain a G All 3 reads have quality 20 99 confidence at this position We want to calculate the p value for calling a G SNP in this column Since the quality values are all equal the p value is the probability of seeing at least 2 G s if there isn t
24. iad 200 400 600 800 1 000 1 200 1 400 1 612 i i i 1 1 i i Consensus ad AS Ce FWD 2 Frag i u D REV 3 Frag a 1 1 a 24 e 6 0 9 0 2 ue Figure 10 5 The overview of a contig Viewing Contigs of Paired Reads In order to view a contig of paired reads you first need to have set up the paired data before assembling see 10 1 4 Once you have your paired read assembly the contig viewer adds an option to Link paired reads in the advanced section of the controls on the right This means that pairs of reads will be laid out in the same row with a horizontal line connecting them Reads separated by more than 3 times their expected distance are not linked by default unless the Link distant reads setting is turned on The horizontal line between paired reads is colored according to how close the separation be tween the reads is to their expected separation Green indicates they are correct yellow and blue indicate under or over their expected separation and red indicates the reads are incorrectly orientated The reads themselves can also be configured to be colored in this way if you use the Paired Distance color scheme from the general top section in the controls on the right settings The colors used and the sensitivity for deciding if reads are close enough to their expected distance can be configured from the Options link when the Paired Distance color scheme is selected You can hover the mouse o
25. select the sequences or alignment you wish to align select the Align Assemble button from the Toolbar and choose Align Whole Genomes An alignment produced by Mauve is displayed in the Mauve genome alignment viewer which allows you to easy see aligned blocks of sequence and genome rearrangements Note that this is not a regular Geneious alignment document and you cannot run downstream analyses such as tree building from this document To run downstream analyses you must first extract the local alignment blocks To do this switch to the Alignment View tab above the sequence viewer and if you have more than one local alignment block choose which one you wish to extract in the General tab to the right of the sequence viewer Then select all the sequences in that alignment and click Extract Choose the option to extract the sequences as an alignment and a separate alignment document will be created in the document table For more information on Mauve and its options please refer to the original documentation for the program http darlinglab org mauve mauve html 118 CHAPTER 9 SEQUENCE ALIGNMENTS LASTZ LASTZ is designed for pairwise alignments of whole genomes and can efficiently align chro mosomal or genomic sequences millions of nucleotides in length To use LASTZ you must first download the plugin by going to Plugins under the Tools menu and selecting LASTZ from the list of available plugins Click the Install button to install it and
26. 2 Alignment graphs In addition to the basic graphs available for individual sequences the following graphs are available for alignments and assemblies Coverage The height of the graph at each position represents the number of sequences which have a non gap character at that position The coverage graph is made up of three bar graphs overlaid on each other a blue graph shows the minimum coverage a black graph shows the mean coverage and a yellow graph underneath the blue and black graphs shows the max imum coverage The minimum graph is drawn over the top of the mean color graph but if necessary the minimum color graph will be reduced in height so that a single pixel of the mean color graph is always visible at each position Thus for sequences which are zoomed in so that the horizontal width of each site is one pixel or more then the graph will be shown in blue with a black line across the top denoting the coverage at that position For large alignments which are zoomed out so that the horizontal width of each site is less than one pixel i e each pixel represents more than one site in the alignment all three bars are visible showing the minimum mean and maximum coverage of bases within that pixel see Figure 9 5 To highlight regions above or below a particular coverage level check Highlight above or Highlight below and a bar will appear below the coverage graph across regions which fit these criteria The Highlight above
27. A Cc 6 0 O Customize Toolbar Back Up M Back QAABAQAQRA Y Name 4 DCN_F primer DCN_R primer DCN gene house mouse gene house mouse gene 2 Insert Sequence Plasmid Vector Possum PopSet pygmy chimpanzee WNT1 gene w History M Forward 3 Connect Disconnect Jabber Description Foward primer for Reverse primer for Homo sapiens decor l Mus musculus 1 Cys Mus musculus 1 Cys An insert sequence Shuttle vector pmMQ91 Genetic structure of tl Pan paniscus clones WNT1 wingless type a New Sequence ica Import Export Divider 45 44 Search we Sequence Search 4 a Agents Do Not Disturb Mode Cancel Cox Figure 2 5 The Toolbar 27 The toolbar can be customized by right clicking Ctrl click on Mac OS X on it This gives a popup menu with the following options e Show Labels Turns the text labels on or off e Large Icons Switches between large and small icons e Customize Lists all available toolbar buttons Selecting deselecting buttons will show hide the buttons in the toolbar 2 5 1 Status bar Below the Toolbar there is a grey status bar This bar displays the status of the currently selected service For example when you are running a search it displays the number of matches and the time remaining for the search to finish 2 6 Geneious menu bar options All of the functions in Geneious can be accessed from the menu bar above the Toolbar This
28. A Jabber ID looks like an email address and has a similar function It uniquely identifies some other Geneious users account You can enter a contact s Jabber ID directly into this field if you know it To see your own Jabber ID hover your mouse over your account in the Sources Panel and it will appear in a tool tip 7 Add Contact to myaccount Jabber ID e g user name talk geneious com 2 Search For Contact Figure 19 3 Add Contact dialog box If the server supports it you should also see a Search For Contact link This opens a search box with some checkboxes indicating what you are searching on Enter all or part of the name or email of the contact you want and click the Search button If any rows are returned in the results table you will be able to select one or entries and add them as contacts Your new contact will appear immediately in your contact list however you will not be able 19 2 MANAGING YOUR CONTACTS 225 3 Add Contact to myaccount Username Name vV Email A Enter Contact ID Add selecte cts Figure 19 4 Add New Contact dialog box in searching mode to tell whether your new contact is online until they accept you as a contact Similarly you may occasionally see a dialog box pop up asking you Allow user name talk geneious com as contact This is another Geneious user attempting to add you as a contact in this manner Your contact will appear grey in your contact list when they are offli
29. Custom Icon Share read only with other Shared Database users db Add Step ete St v Edit Optio Help A Alignment gt MAFFT Alignment P TreeBuilding gt MrBayes Optic landom Seed 824 Figure 16 2 The Edit Workflow window Add Operation from 97 available Add Recently Used Operation gt For Each Document For Each Sequence Extract Sequences From List T Group Documents TF Group Sequences Add document chosen when running workflow Combine With Earlier Document s Save Documents Branch Rename Document s L Copy Property Between Documents 4 Filter Documents F Sort Documents 2 Custom Java Code Figure 16 3 Add step Menu options for building and editing Workflows selected from your Geneious database For example a single document can be an alignment a sequence list a tree or a stand alone sequence The Add Step button Figure 16 3 provides a dropdown menu with a range of Steps that can be added to your WorkFlow The purpose function of each type of Step is summarised in Table 16 1 16 2 1 Configuring options for Operation and Steps For each Operation added to your Workflow you can edit and specify values for the config urable options available for each operation To do this select the Step you wish to configure and click View Edit Options To set the Workflow up so that the options are preconfigured and cannot be changed when the workflow is run select Expose no options
30. Frag AGGAGG AACACCGGT Ao YA OY AAATAACTGACGCTGAGG A A A ZA AKALAN SKALA Ce REV 3 Frag AGGAGGAACACCGG TGGCGAAGGCGI JGT CTCTGGGAAA TAACTGACGCTGAGG Figure 10 6 Highlight disagreements and edit to resolve them 10 6 Extracting the Consensus Once you are satisfied with a contig you can save the consensus as a new sequence by clicking on the name of the consensus sequence in your contig and clicking the Extract button You can also generate consensus sequences for single or multiple contig documents by selecting the documents and going to Tools Generate Consensus Sequence 144 CHAPTER 10 ASSEMBLY AND MAPPING Chapter 11 Analysis of assemblies and alignments 11 1 Finding polymorphisms To easily identify bases which do not match the consensus turn on Highlighting in the con sensus section of the sequence viewer options Select the options Disagreements to Consensus this is the default setting for nucleotide alignments When this is on any base in the se quences which matches the consensus at that position is grayed out and bases not matching are left colored With this on you can quickly jump to each disagreement by pressing Ctrl D command D on Mac OS X or by clicking the Next Disagreement button in the sequence viewer option panel to the right Each disagreement can then be examined or resolved You can also use this feature if you have aligned to a referenc
31. Pongo abelii isolate Orang_5 cytochrome 1 684 1 198 72 0 99 4 98 8 U12704 Pongo abelii Ppy3 cytochrome oxidase s 1 684 1 198 72 0 99 4 98 8 EU835092 Pongo abelii isolate Orang_32 cytochrom 1 684 1 195 12 0 99 4 98 8 EU835091 Pongo abelii isolate Orang_1 cytochrome 1 683 1 198 72 0 99 4 98 8 EU835090 Pongo abelii isolate Orang_4 cytochrome 1 684 1 198 72 0 99 4 98 8 EU835082 Pongo abelii isolate Orang_14 cytochrom 1 684 1 198 72 0 99 4 98 8 EU835081 Pongo abelii isolate Orang_25 cytochrom 1 684 1 198 72 0 99 4 98 8 EU835080 Pongo abelii isolate Orang_16 cytochrom 1 684 1 193 31 0 99 3 98 7 EU835079 Pongo abelii isolate Orang_17 cytochrom 1 684 1 193 31 0 99 3 98 7 EU835078 Pongo abelii isolate Orang_22 cytochrom 1 684 1 191 51 0 99 3 98 6 EU835095 Pongo abelii isolate Orang_15 cytochrom 1 684 1 178 89 0 99 2 98 3 EU835093 Pongo abelii isolate Orang_2 cytochrome 1 684 Hide Selected sequences are only summaries Download Full Sequence s Dotplot Dotplot Self DNA Fold Distances Text View Download Info BD Cr extract GRC WM Translate di Add Edit Annotation 24 Allow Editing Annotate amp Predict ave 9080 ao Consensus i i i i i i identity e Ce 1 Orangut 2 X97707 Erkens Alt click on a sequence position or annotation or select a region to zoom in Alt shift click to zoom out Figure 15 2 BLAST Complete You can also download the full da
32. Shared Databases will work with another database system if you provide its driver but it is likely that it will To supply your own driver open up the dialog you would normally use to connect to a database Then click the More Options button 20 3 REMOVING A SHARED DATABASE 231 20 3 Removing a Shared Database To remove a Shared Database simply right click on its top folder and choose Remove database 20 4 Administration The typical user will not have to do any administration this section is for those in charge of the database 20 4 1 Groups and Roles Shared Databases support user groups and roles for managing access to documents This means that you can restrict access of folders to privileged people How it works is that each folder in Geneious belongs to a group Users can belong to any number of groups and have a specified role within that group The three roles are e View allows the user to view the contents of folders e Edit allows the user to view and edit the contents of folders e Admin allows the user special administrative functions on folders As of this time Geneious only uses the Admin role for the Everybody group By default there is only one group the Everybody group When a user logs in for the first time Geneious will put them into the Everybody group with a role of Edit So this means every user of the Shared Database belongs to this group with a role of Edit unless you enter them into the g_u
33. View Pressing the View Descendants button will bring up this view Figure 6 4 E Descendants of first nee first E B Ligate Sequences Today at 1 56 PM pg Ligated sequences Figure 6 4 Descendants view When you choose to begin editing a document with actively linked parents in the Sequence View you will immediately be warned that in order to save your changes you will need to deactivate this link As with the Actively Linked Descendants view you will be given the op portunity to view the document s lineage Editing a document that is a descendant of other documents is usually unintentional however in some circumstances you may simply be inter ested in the output documents of an operation not the parent descendant relationship and as such you may hide this dialog Figure 6 5 88 CHAPTER 6 PARENT DESCENDANT TRACKING Actively Linked Parents Ea This document has an actively linked parent View Parents If you edit it and save your changes you will need to either deactivate the link s or save your changes to a copy Continue Editing Cancel Figure 6 5 Actively Linked Parents dialog Upon conclusion of your editing you will again be prompted to either deactivate links or save a copy 6 2 The Lineage View Every document that is linked actively or otherwise to another document has a tab called Lin eage in the Info View tab The lineage view allows you view parent desce
34. a column in the document table and can be treated as a user defined field for use in sorting searching and filtering your documents When multiple documents are selected the Properties view displays all of the fields and meta data belonging to the selected documents When all documents have the same value for a field it is displayed in the viewer If the documents have different values or some of the selected documents do not have a value then the field will show that it represents multiple values Changes made to the fields will apply to all selected documents You can add meta data to any of your local documents including molecular sequences phy logenetic trees and journal articles You cannot add meta data to search results from NCBI or EMBL etc until the documents are copied into one of your local folders 5 7 1 Adding meta data To add meta data to your document select the Add Meta Data button on the toolbar and then choose from the available types Selecting a meta data type will create an empty instance of that type To fill meta data values just start typing into the fields See section 5 7 3 for information on how to create a new meta data type 5 7 2 Editing Meta Data To edit existing meta data fields simply click on the field and enter your data Some fields may have constraints which you can edit in the Edit Meta Data Types dialog see 5 7 4 If the data you have entered does not conform to the constraints of the field
35. a value for the column e Tracks allows you to specify what tracks are displayed in the table e Columns allows control over which columns are visible in the table To further filter what is visible in the table use the filter box at the top right of the table Filtering is only done against the currently visible columns for each annotation To export the visible rows and columns of your annotations table click Export table This exports the table to a CSV comma separated values file The Extract and Translate buttons will create a new document from the selected annotation s Extract extracts the region of the selected annotation to a new document Translate translates the nucleotides in the region of the selected annotation into amino acids using your choice of reading frame and genetic code and saves it to a new protein document Annotations table functions can also be accessed via a popup menu when right clicking on one or more selected annotations in the table This menu contains options for copying the selected value extracting translating showing on sequence editing and deleting the selected annotations The show on sequence function in this list will show the selected annotations in the sequence viewer 8 1 3 Editing annotations Annotations can be edited by selecting them either on the sequence or from the annotations table and clicking Edit Annotations This brings up a window where the annotation name type location p
36. allows you to choose the options for how your consensus sequence 124 CHAPTER 9 SEQUENCE ALIGNMENTS is called as described above and then saves it to a separate document If your consensus sequence contains characters where there are regions of no low coverage in your assembly you can split the consensus sequence at these bases to generate multiple sequences by checking the option to Split into separate sequences around calls Chapter 10 Assembly and Mapping Assembly is normally used to merge overlapping fragments of a DNA sequence into a contig which can be used to determine the original sequence The contig essentially appears as a mul tiple sequence alignment of the fragments After some manual editing of the contig to resolve disagreements between fragments which result from read errors the consensus sequence of the contig is extracted as the sequence being reconstructed Contig assembly is also used to align a large number of reads of the same sequence from different individuals This is done to find small differences between reads or SNPs Single Nucleotide Polymorphisms In this type of analysis the consensus sequence of the contig is not the interesting part the differences between fragments is This can also be done against a known reference sequence when differences between each of the fragments and the reference are of interest 10 1 Read processing 10 1 1 Trim Ends Trimming low quality ends of sequences is norma
37. an effective recognition site of at least 4 bases and where the resulting overhang is at least 3 bases on any strand Usually the receiving type IIS sites already present in your backbone 14 6 GOLDEN GATE 193 will define the site you will use these do not have to be annotated on the backbone Note that the Geneious Golden gate tool currently does not allow the use of multiple type IIS restriction enzymes for assembly Each sequence including the backbone is represented by a tag in the inserts field with relevant information shown on the tag or next to it If all overhangs are compatible the backbone will be shown in green and the inserts in grey Inserts or backbones with incompatible overhangs are shown in red The information shown on the tag for each line is as follows e Sequence Name highlighted in blue if reverse complemented e Used reactions Pre digested if the sequence has existing overhangs on both ends Cut by enzyme if the sequence will be cut by two enzymes PCR Product if restriction sites for the chosen enzyme will be introduced via PCR and at least one of the existing primers on the sequence is used Full length PCR if newly generated primers will amplify the full sequence and ap pend restriction sites at both ends Overhang Enzyme or Primer for either side if a mix of either reaction is used If conflicts have been detected a warning will be shown and the tag will be high lighted in red
38. and annotations as described in the previous option If multiple input sequences are selected the result sequences are placed in a single sequence list document Enter the name of the new document in the text input box of this option Source Genetic Code lets you select the genetic code to be used when translating the source sequences If you have selected multiple source sequence documents with dif ferent genetic codes the choice Multiple Values will be available to indicate that the genetic code associated with each document should be used You can select a genetic code other that the one that is shown as the default for the selected input documents if you want to override the default Target Organism lets you select a Codon Usage Table from EMBOSS for the target ex pression host Each table contains the codon usage statistics for a selected set of genes of the organism Target Genetic Code lets you specify the genetic code of the target organism The EM BOSS CUT file format that is used for the Target Organism codon usage data does not contain a field for the genetic code You must select the correct genetic code for the target organism that you select Threshold to be rare lets you specify the relative adaptiveness value W for a codon below which the codon is considered to be rare and will be replaced with the highest value synonymous codon Set the threshold to 1 to change all codons that are not the highest value in their
39. bar at the bottom of the tree view to indicate the length of the branches of the tree It has three options Scale range font size and line weight Setting the scale range to 0 0 allows the scale bar to choose its own length otherwise it will be the length that you specify 12 5 VIEWING AND FORMATTING TREES 163 12 5 8 Statistics Displays information on the number of nodes and number of tips in the tree 12 5 9 The Toolbar The buttons on the toolbar along the top of the viewer allow you to edit the tree Click on a node in the tree viewer to select the node and its clade Double click the node to collapse un collapse the clade in the view Once you have selected a clade in the view you can edit it using the following toolbar buttons Color Nodes allows you to choose a new color for the selected clade Font allows you to change the font for the tip labels e Root allows you to re root the tree on the selected node e Swap Siblings allows you to swap the position of the sibling clades of the selected node In version 9 onwards the toolbar also contains a Search box that allows you to search for particular tip labels If a match is found this tip is displayed on the tree and all other tips are greyed out If you wish to search by a field that is not currently displayed on a tip label you need to change the field under Show Tip Labels first 164 CHAPTER 12 BUILDING PHYLOGENETIC TREES Chapter 13 PCR Primers Geneious
40. circular sequences to be shown linearly e Spaces every 10 bases If you are zoomed in far enough to be able to see individual residues then an extra white space can be seen every 10 or whatever number you choose residues when this option is selected Properties 72 CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES e Numbering Enables the display of base position number above the sequence residues For alignments and assemblies options are available for displaying the numbering of consensus reference alignment and or all original sequences e Mini map Enables the display of a mini map at the top of the sequence viewer which highlights the currently displayed location in the entire sequence e Outline residues when zoomed out This adds a fine line around the sequence which can help with clarity and printing Annotations e Labels This option changes where the labels are displayed on the annotation Inside Outside Inside or Outside and None e Overlay when zoomed out When only a single annotation covers a region it will be placed on top of the sequence e Compress annotations This option reduces the vertical height of the annotations on dis play This reduces the space occupied by annotations by allowing them to overlap and increases the amount of the sequence displayed on the screen e Show arrow tips Displays the directional indicator for an annotation as a large arrow tip e Hide excessiv
41. details For each result from the previous Operation combines with the cor responding input document s of an earlier operation in the Work flow The documents from the earlier Operation are added to the end of the list of documents from the previous operation Optionally saves the current results Also optionally starts a branch beginning from an earlier Step in the Workflow Rename Documents 2 Copy Property Between Documents 47 Filter Documents F Sort Documents Renames the document s Works on a pair of documents Copies a property from one docu ment to another document and outputs just the destination docu ment If only a single document is provided outputs that document unmodified Filters some documents based on the content of their fields Sorts some documents based on the contents of their fields Custom Java Code Write some custom Java code to do whatever you want See section 16 3 for more details 16 2 CREATING AND EDITING WORKFLOWS 215 Operation options you want the workflow to use To allow some or all or the options to be configured each time the workflow is run select Expose all options or Expose some options Exposed options can be presented in a number of ways as described below Optionally label exposed options as Use this to group and label all the exposed options for this Workflow Step under a labelled section rather than mixing the options for this Step in with options from oth
42. do in your contact s own Sources panel You can browse these folders as you do your local folders You can also search a shared folder just as you can a local one Additionally you can search all of a contact s shared documents by clicking on the contact itself and then conducting the search You can also search all the shared documents of all of an account s contacts by clicking on the account and conducting the search Agents can be set up on shared folders contacts and accounts You cannot search browse or run or set up agents on a contact that is currently offline When you first view your contact s documents in the Document Table the documents you see are only summaries To view the whole document select the summary s of the documents s you would like to view and the click the Download button inside the document view or just above it Download options are also available in the File menu and in the popup menu when document summary is right clicked Ctrl click on Mac OS X The size of these files is not displayed in the Documents Table You can cancel the download of document summaries by selecting Cancel Downloads from any of the locations mentioned above 19 5 CHAT 227 19 5 Chat You can either chat with a single contact or invite several contacts to join you in a new chat 19 5 1 Chatting with One Contact To start chatting with a particular contact who may be online using Geneious or another chat client which uses the Jabbe
43. dragging To pan hold shift then double click and drag YYYY YY YYY vY v Y Figure 7 2 A view of a 3D protein structure in Geneious Highlight El Select 93 94 CHAPTER 7 RNA DNA AND PROTEIN STRUCTURE VIEWER The select button lets you select all none or the nonselected region of the structure as well as by element group type or secondary structure The highlight selected checkbox lets you select whether to highlight the selected atoms in the structure view The structure tree shows the atoms in the structure in a tree format Click on regions in the tree to select those regions You can also Shift click and Ctrl click to select multiple regions at once The command box lets you type in arbitrary jmol scripting commands To see some exam ples select one of the pre populated options in the box s drop down For a complete de scription of the commands you can use see http www stolaf edu academics chemapps jmol docs 7 2 3 Display Menu At the top of the viewer is the display menu Here you can modify the appearance of the structure Reset lets you reset the position of the structure reset the appearance of the structure to the default or reset the appearance of the structure to its appearance when it was last saved Color lets you change the color scheme of the selected region of the atom Style lets you change the style of the selected region of the molecule eg to spacefill or cartoon view Atoms l
44. in brackets When the Local folder or a sub folder is collapsed minimized the brackets next to the folder shows how many files are contained in that folder as well as all of its sub folders In addition if some of the documents in a folder are unread the number of unread documents will also appear in the brackets The document types available are listed in Table 4 1 To create a new folder in Geneious select the Local folder or a sub folder icon in the Sources panel and either right click Ctrl click on Mac OS X and select New folder from the popup menu or go to File New Folder This will open a dialog where you can name your new folder This folder will then be created within the folder you originally selected You can also delete rename move export or change the color of a folder by right clicking on the folder or control click on MacOSX and selecting the option you require from the menu These options are also available under the File menu In Mac OS X you can also use the plus and minus buttons located at the bottom of the service panel to create and delete folders 4 1 1 Moving files around Files can be moved between folders in a number of ways Drag and drop This is quickest and easiest Select the documents that you want to move Then while holding the mouse button down drag them over to the desired folder and release If you 51 52 CHAPTER 4 MANAGING YOUR LOCAL DOCUMENTS Table 4 1 Geneious document t
45. in that format as long as no changes have been made to the document Both fasta and fastq files can be exported in compressed fasta gz and fastq gz format for smaller file size If exporting paired reads in fasta format an option to export the forward and reverse reads to separate files is available by choosing Fasta Paired Files as the file type option 3 5 1 Export to comma separated CSV or tab separated TSV files The values displayed in the document table can be exported to a csv file which can be loaded by most spread sheet programs When choosing to export in csv format Geneious will also present a list of the available columns in the table including hidden ones so you can choose which to export 3 6 PRINTING AND SAVING IMAGES 49 3 5 2 Exporting multiple files There are several options for export of multiple files from Geneious e Export to a single file Multiple files can be exported to a single file by selecting all the files you wish to export and going to File Export Selected Documents This will combine all the files you selected into a single file for export Batch Export This option exports each selected file as a different document E g you can select several sequence documents and use Batch Export to export each sequence as an individual fasta file The options for batch export let you specify the format and folder to export to as well as the extension to use Each file will be named according to th
46. it will be displayed in red 82 CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES Alignment View Distances Text View x La EZ Add Meta Data E Save eg OI a Properties Name People id Description Multiple alignment of 5 sequences from fictitious characters Lineage Notes Source x Depth 15 LatLong Date Collected 12 Sep 2012 Click Add Meta Data in the toolbar to add your own custom information Figure 5 7 The Properties View and you will be shown the field s constraints in a tooltip Tip To enter a new line in a text field press Shift enter or Ctrl enter 5 7 3 Creating a new Meta Data type Geneious does not restrict you to the meta data types that it comes with You can create your own types to store any information you want To create a new type click Edit meta data types then click the Create button at the bottom left of the panel This creates a new type with one empty field and displays it in the panel to the right Note The Name and Description fields distinguish your meta data type from other user defined types They do not have any constraints Next you need to decide what values your Meta Data Type will store by specifying its fields 5 7 META DATA 83 Field name This defines what the field will be called It will be displayed alongside columns such as Description and Creation Date in the Documents Table You can have more than one Field in a single Meta
47. method of multiple sequence alignment is progressive pairwise alignment The idea is very simple At each step a pairwise alignment is performed In the first step two sequences are selected and aligned The pairwise alignment is added to the mix and the two sequences are removed In subsequent steps one of three things can happen e Another pair of sequences is aligned e A sequence is aligned with one of the intermediate alignments e A pair of intermediate alignments is aligned This process is repeated until a single alignment containing all of the sequences remains Feng amp Doolittle were the first to describe progressive pairwise alignment Their algorithm used a guide tree to choose which pair of sequences alignments to align at each step Many variations of the progressive pairwise alignment algorithm exist including the one used in the popular alignment software ClustalX Multiple sequence alignment in Geneious To run a multiple alignment in Genieous select all the sequences you wish to align and click Align Assemble Multiple align Select Geneious as the alignment algorithm The Geneious 9 2 SEQUENCE ALIGNMENTS 115 multiple alignment algorithm uses progressive pairwise alignment The neighbor joining method of tree building is used to create the guide tree As progressive pairwise alignment proceeds via a series of pairwise alignments this function in Geneious has all the standard pairwise alignment options In addit
48. now select the Create or Create and Run The agent will then be added to the list in the agent dialog and it will perform its first search if you clicked Create and Run Otherwise it will wait until its next scheduled search All downloaded files are stored in the destination folder and are marked unread until viewed for the first time 3 4 2 Checking agents Once you have created one or more agents Geneious allows you to quickly view their status in the agents window which is accessible from the toolbar Your agents details are presented in several columns Enable Action Status and Deliver To Enable This column contains a check box showing whether the agent is enabled Action This summarizes the user defined search criteria It contains 46 CHAPTER 3 IMPORTING AND EXPORTING DATA Search Database PubMed Match all of the following contains Drummond Deliver To Local 0 n Search every 1 _ Make destination folder a smart folder _ Only get documents created after today cancel Greate Gresteand Ron Figure 3 3 The Create Agent Dialog 3 4 AGENTS 47 1 Details of the database accessed For example Nucleotide and Genome under NCBI 2 The search type the Agent performed e g keyword 3 The words the user entered in the search field for the Agent to match against Status This indicates what the Agent is currently doing The status will be one of the fol
49. one go Workflows shared by other users in a server database are indicated by an amp icon These Work flows will not appear in the drop down menu unless you choose to show them Workflows 211 Workflows Y Align DNA then build tree 7 Align DNA via Muscle ClustalW and Geneious E Y 7 Batch alignment with MUSCLE AC Batch Restriction Cloning g ag Batch reverse complement Z I Combined mapping and de novo assembly g Extract Sequences By Name A gt Filter Y A Identify Organism E Z O List all sequences with variants f Z Map reads then find variations SNPs CHAPTER 16 WORKFLOWS New Workflow 2P View Edit ii Copy XK Delete J Hide from Drop Down List Y Z Map reads to each reference sequence Move Up Y ZZ Merge mapped sequences Y amp Sequence Search Align and Build Tree ceci Y A Set CDS Translation Property g O Shift circular origin to specified nucleotides Export 8 KS Split Sequence List E Ade Trim and Filter E5 Import g G Truncate Annotations Help 4 Align DNA twith MAFFT then build tree with Mr Bayes E Y Show sample workflows 3 MV Show shared workflows OK Figure 16 1 The Manage Workflows window shared by other users in a shared database are only editable by the creator However you can use the Copy button to create your own personal editable copy of any Workflow 16 2 Creating and editing Workflows To create or edit a wor
50. phisms on any track Then choose the other annotation type e g CDS for Set B Uncheck Names must match as in general polymorphism annotation names do not match those of other annotation types and check Allow intervals to partially match and produce partial annota tions Under Results choose to return A amp B annotations common to both sets This will return a new track containing annotations of the type in Set A polymorphisms that are con tained within Set B CDS annotations See Figure 8 6 106 CHAPTER 8 WORKING WITH ANNOTATIONS Example 2 finding polymorphisms in a child which are not present in either parent To return polymorphisms which are unique to the offspring set Annotation Type as Polymor phism for Sets A B and C and set A and B as the parent tracks and C as the child track as in Figure 8 7 Check Names must match and Allow intervals to partially match and produce par tial annotations Choose C A B in the results display This will return a new track containing annotations found in Set C child but not in Sets A and B parents 8 3 COMPARE ANNOTATIONS Annotation Types Set B Annotation type CDS 7 anywhere Set A Annotation type Polymorphism anywhere Set C Annotation type gene ontrack son Comparison C Names must match _ All properties must match Y Allow intervals to partially match and produce Partial Annotations Results A B
51. primers between two inserts who share half of the specified overlap length each For very short or long extensions primer3 might fail to calculate a Tm If the sequence is too short Formula 14 1 is used if it has a length greater than 36bp Geneious uses the Formula 14 2 Tm 2 AT 4 GC 14 1 14 5 GATEWAY CLONING 191 GC 16 4 ATGO Tm 64 9 41 14 2 A Report Document will be generated listing the generated products and primers in a tabular view Errors that occurred during the primer generation process will be reflected in that re port document Furthermore any modifications recession or maintaining overhangs adding extensions to primers are shown at the beginning of the document Geneious has a built in parent descendant tracking system Whenever a change is made to a parent sequence it will ask to propagate this change to its offsprings However in the case that the user introduced some changes in the primer binding region or the extension region of the original sequences these changes won t be reflected in the report document 14 5 Gateway Cloning Geneious contains three operations to assist with Gateway cloning Gateway is a registered trademark of Invitrogen Corporation The Gateway option under the Cloning menu will per form a BP reaction and or an LR reaction on the selected documents If there are a mixture of AttB AttP and AttL AttR sites on the input documents it will perform a BP reaction on
52. primers will be designed to the very ends of this region so that the entire region is included in the PCR product This option is useful for amplifying an entire CDS for creating an insert for cloning 13 1 3 Tm calculation This section gives the formulas used to calculate the melting point of oligos Under Formula you can choose between two different tables of thermodynamic parameters and methods for melting temperature calculation e Breslauer et al 1986 This is used by old versions of Primer3 until version 1 0 1 and uses the formula for melting temperature calculation suggested by Rychlik et al 1990 e SantaLucia 1998 This is the recommended value Three different Salt Correction Formula options are available e Schildkraut and Lifson 1965 This is used by old versions of Primer3 until version 1 0 1 e SantaLucia 1998 This is the recommended value e Owczarzy et al 2004 168 CHAPTER 13 PCR PRIMERS 13 1 4 Characteristics The Characteristics section allows you to set absolute limits on properties of primers and probes such as melting point and GC content Optimum values can also be specified For details on individual options hover your mouse over them and a popup box will describe the function of the option Characteristics can be set for either Primers or DNA Probes depending on the task you have chosen The Primer section is available if one of Forward Primer or Reverse Primer is being designed or tested and DNA Pr
53. range of a complementary pair will be returned You can specify the maximum overlap of the paired sites and the max imum space allowed between the paired sites The maximum overlap and maximum space between sites are measured from the 5 PAM distal end of the CRISPR sites The optimal CRISPR pairs will be linked when they are annotated onto your target sequence though any CRISPR site with a potential pair will be returned Optimal pairs are decided by averaging their off target interaction scores Results output CRISPR sites are returned as annotations on your original sequence Hovering the mouse over an annotation will bring up a tooltip showing the information about any scores calculated plus the 5 highest scoring off target sites if they are analysed For each off target site the score location and off target sequence are given Mismatches to the CRISPR site are in red and insertions in the off target site are underlined and red The annotations are colored based on their On target Off target or Paired score If more than one type of score is calculated you can choose which score to color the annotations by using the Color CRISPR Sites by option The annotations will be colored on a gradient starting at green for good scores moving through yellow and ending at red for poor scores 14 9 Optimize Codons The option Optimize Codons from the Annotate amp Predict menu allows you to adapt a nucleotide sequence to the genetic
54. reference sequence Mixture multiple variations from the reference sequence which are not all the same length For variations inside coding regions CDS annotations the following fields can be displayed 148 CHAPTER 11 ANALYSIS OF ASSEMBLIES AND ALIGNMENTS e Codon Change indicates the change in codon Essentially this is the same as the Change field but extended to include the full codon s For example TTC TTA e Amino Acid Change indicates the change if any in the amino acid s by translating the codon change For example F L Protein Effect summarizes the change on the protein as either a substitution frame shift truncation stop codon introduced or extension stop codon lost 11 2 Analyzing Expression Levels 11 2 1 Calculating Expression Levels The Calculate Expression Levels feature from the Annotate amp Predict menu calculates nor malised expression measures from mapped RNA seq data RPKM FPKM and TPM are cal culated for each CDS annotation on the reference sequence of a contig and the results are dis played as a heat map annotation track If you have multiple reference sequences for each sample e g reads mapped to multiple chro mosomes all contigs from a single sample should be selected and run ina single step To calculate differential expression between samples you need to run Calculate Expression Levels for each sample separately and then compare the results using Compare Expr
55. right when you insert sequence to get them to shift to the left hold down the Shift key as you insert the sequence To overwrite sequence select the sequence you wish to overwrite and type or paste the new sequence in 76 CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES To select sequences or regions of sequence there are several options To quickly select a single residue double click on it To select a block of residues within a single sequence triple click To select a block of residues across multiple sequences quadruple click To select a block of 10 residues hold down Shift and Cntrl alt option on a Mac and press the keyboard arrow To select a specific region of sequence click at the beginning of the region you want to select hold down Shift and then click the end To select the same region across multiple sequences in an alignment select the region you want in the first sequence then hold down Shift alt command on a mac and click the down arrow to apply the selection to the sequences underneath Holding down Shift Cntrl alt option on a mac while pressing the down arrow will select the sequences in batches of 10 Sequences can be reordered within an alignment by clicking the sequence name and dragging Sequences can be removed from an alignment by right clicking Ctrl click on Mac OS X on the sequence name and choosing the remove sequence option Alternatively select the entire sequence by clicking on th
56. seq2 YEGLTTANGXKEY YODKNGGNFFKLREDWWTANRETVWKAITCGA seg3 KRIYKKIFKEIHSGLSTKNGVKDRYQN DGDNYFQLREDWWTANRSTVWKALTCSD seg4 SORHYKD DGGNYFOLREDWWTANRHTVWEAITCSA seg NVAALKTRYEK DGONFYOLREDWWTANRATIWEAITCSA seg FSKNIX OQIEELODEWLLEARYKD TDNYYELREAWWTENRHTVWEALTCEA seq7 KELWEALTCSR segl GGGKYERNTCDG GONPTETONNCRCIG ATVPTYFDYVPOYLRWSDE seq2 P GDASYFHATCDSGDGRGGAQAPHKCORODG ANVVPTYFDYVPOFLRWPEE seg3 KLSNASYFRATC SDGOQSGAQANNYCRCNGDKPDDDKP NTDPPTYFDYVPQYLRWSEE seq4 DKGNA YFRRTCNSADGKSQSQARNOCRC KDENGKN ADOVPTYFDYVPQYLRWSEE seg5 DKGNA YFRATCNSADGKSOSQARNOCRC KDENGXN ADOVPTYFDYVPQOYLRWSEE seg6 P GNAQYFRNACS EGKTATKGKCRCISGDP PTYFDYVPOYLRWSEE seg P KGANYFVYKLD RPKFSSDRCGHNYNGDP LTNLDYVPOYLRWSDE CSFASTA format ABI csfasta files represent the color calls generated by the SOLiD sequencing system DNAStar sequences DNAStar seq and pro files are used in Lasergene a sequence analysis tool produced by DNAStar DNA Strider sequences Sequence files generated by the Mac program DNA Strider containing one Nucleotide or Pro tein sequence EMBL Swiss Prot sequences Nucleotide sequences from the EMBL Nucleotide Sequence Database and protein sequences from UniProt the Universal Protein Resource 36 CHAPTER 3 IMPORTING AND EXPORTING DATA EndNote 8 0 9 0 XML EndNote is a popular reference and bibliography manager EndNote lets you search for journal article
57. sequence data each sequence can be annotated with descriptive sequence information CSV TSV Comma Tab Separated Values sequences Sequences such as primer lists are often stored in spreadsheets Geneious has an importer that can be given the field values for a spreadsheet file exported in CSV or TSV format and it will import them and convert them to documents as well as preserving the additional field contents It can handle nucleotide and amino acid sequences as well as primers and probes For more information on importing primers from a spreadsheet see the PCR Primers section 40 CHAPTER 3 IMPORTING AND EXPORTING DATA SAM BAM alignment SAM and BAM format are produced and used by SAMtools SAM BAM files contain the results of an assembly in the form of reads and their mappings to reference sequences Chromatograms Sequence chromatogram documents contain the results of a sequencing run the trace and a guess at the sequence data base calling Informally the trace is a graph showing the concentration of each nucleotide against sequence positions Base calling software detects peaks in the four traces and assigns the most probable base at more or less even intervals VCF variant calls The VCF format contains sequence annotation information You can use a VCF file to anno tate existing sequences in your local database import entirely new sequences or import the annotations onto blank sequences Vector NTI Geneious su
58. sequence prediction and annotation will also appear in this menu when installed e Trim Ends Trims vectors primers and or poor quality sequence see section 10 1 1 e Annotate from Database Annotates sequences with similar annotations from your database See section 8 2 3 e Find ORFs Finds all open reading frames in a sequence and annotates them e Search for Motifs Searches for motifs in PROSITE format Uses fuzznuc and fuz zpro from EMBOSS e Find Variations SNPs Finds variable positions in assemblies and alignments See section 11 1 1 e Find Low High Coverage Finds regions with low or high read coverage in assemblies See section 10 4 e Calculate Expression Levels Calculates expression levels RPKM FPKM TPM for a single sample See section 11 2 e Compare Expression Levels Compares expression levels between two samples See section 11 2 e Transfer Annotations Copies annotations to the reference and or consensus sequence of an alignment or assembly See section 8 2 3 e Compare Annotations Compares annotations across up to 3 annotation tracks or docu ments See section 8 3 2 6 7 Help Menu This consists of the standard Help options offered by Geneious Help shows and hides the Help panel 32 CHAPTER 2 THE GENEIOUS MAIN WINDOW Tutorial shows and hides the Tutorial panel Online Resources gives access to a variety of resources on our website Check for Updates checks for new versions o
59. sequences in your database and offer them as options in the list of oligo sequences There is no need to select them along with the target sequence before starting the operation The meta data type Primer Info can be used to note the fridge location etc of a particular primer 13 5 TEST WITH SAVED PRIMERS 175 13 5 Test with Saved Primers Primers and probes can also be quickly tested against large numbers of sequences to see which ones the primers will bind to To test primers select the target sequences you want to test for compatibility with primers and choose Primers Test with Saved Primers en Test with Saved Primers te Primer3 if y Test specific primers against the selected sequences m Forward Primer DCN F primer Choose Reverse Primer DCN_R primer Probe None Choose _ Search for saved primers that match the selected sequences vs Region Input Options ve Included Region 1 53 Target Region 1 _ Product Size Between Optimal Product Size _ Maximum Mismatches Mismatch Options gt Tm Calculation 3 Cancel ok Figure 13 6 The primer test dialog There are two ways in which Geneious can test your selection of primers and probes The first option in the dialog tests a specific forward and reverse primer pair and or probe Clicking the Choose buttons next to forward reverse and probe options will bring up your primer database allowing you to select any primer in your database for testing The
60. sizes in the toolhar above Figure 12 3 A view of a phylogenetic tree in Geneious The tabs to the right of the tree viewer contain options for controlling the look of the tree and the information displayed on it The toolbar above the tree provides additional formatting options and allows you to change the root or rearrange the tree The subsequent sections provide more detail on these options 12 5 1 Current Tree If you are viewing a tree set this option will be displayed Select the tree you want to view from the list 12 5 VIEWING AND FORMATTING TREES 161 12 5 2 General The General tab has 3 buttons showing the different possible tree views rooted circular and unrooted The Zoom slider controls the zoom level of the tree while the Expansion slider expands the tree vertically in the rooted layout 12 5 3 Layout This has different options depending on the layout that you select above e Root Length Sets the length of the visible root of the tree Rooted and Circular trees e Curvature Adds curvature to the tree branches Rooted view only e Align Taxon Labels Aligns the tip labels to make viewing a large tree easier Rooted view only e Root Angle Rotates the tree in the viewer Circular and Unrooted views e Angle Range Compresses the branches into an arc Circular view only 12 5 4 Formatting The following options are available for formatting branches e Flip the tree horizontally flips the tree so that branc
61. that they have many large files in their local database preventing Geneious from starting if this value is reset to the normal default 700M on 32 bit 1000M on 64 bit This is an issue because the Info plist file is stored in the Geneious app bundle so it gets replaced when upgrading 22 6 Web Linking to Data in Geneious It is possible to create web links which will open data in Geneious when they are clicked in another program This only works on Windows and Mac OS and Geneious has to be installed on the machine where the link is clicked Some other programs may not support these type of links There are two types of links supported one which will download and import a file into Geneious and one which will select documents which are already stored in Geneious To create a web link that opens Geneious and imports a file from a given location use the following form of URL geneious file lt PathToFile gt eg geneious file http www cambridgeigem org gbdownload pSB1C3 gb will download and import the pSB1C3 vector from the iGEM parts registry The file can be of any format which Geneious is able to import see 3 2 including plugin format Only one file can be linked to in this way 242 CHAPTER 22 ADVANCED ADMINISTRATION To create a web link that opens Geneious and selects a document which is already in your local folders or on a Shared Database use the following form of URL geneious urn lt URNofDocument gt or
62. the most frequently used codon for that organism is used for each amino acid 5 6 VIEWING CHROMATOGRAMS 79 5 6 Viewing chromatograms Geneious can view chromatogram information from files imported in ab1 or scf format If the chromatograms are not visible check Chromatograms under the Graphs tab see Figure 5 6 Chromatogram files are produced from sequencing machines such as the Applied Biosystems 3730 DNA analyzer The raw output of a sequencing machines is known as a trace a graph showing the concentration of each nucleotide against sequence positions The raw trace is pro cessed by a Base Calling software which detects peaks in the four traces and assigns the most probable base at more or less even intervals Base calling may also assign a quality measure for each such call typically in terms of the expected probability of making an erroneous call Geneious does not perform base calling itself this information is already contained in the ab1 or scf file Chromatogram peaks for individual bases can be turned off by checking the A G C T boxes in the Graphs tab Note that since the distance between bases as inferred from the trace varies the trace may be either contracted or expanded compared with the raw data The vertical scale of the chromatogram can be adjusted by clicking and dragging on the graph itself The total height of the graph can be adjusted by increasing the number displayed next to the graph on the right of the Se
63. the sequence viewer or choose Annotate and Predict Annotate From Database from the menu 3 Click on the folder name next to the label Source and in the window that appears select 104 CHAPTER 8 WORKING WITH ANNOTATIONS the folder containing your annotation database then click OK 4 Adjust the similarity setting if necessary 5 If you have any short annotations click on the Advanced button and adjust the Index Sequence Length 6 Click Apply This will apply any matching annotations that have been found in your annotation database to your sequence s If you wish to only apply only some of the annotations to your sequence select the annotations you want either directly on the sequence or in the annotations table before you click Apply This will apply only the selected annotations onto the sequence rather than all of them 8 2 4 Annotation of sequence features using EMBOSS tools The EMBOSS tools which were available under the Annotate and Predict menu in previous Geneious versions are now available as separate plugins EMBOSS protein analysis includes antigenic to predict antigenic regions sigcleave to predict signal cleavage sites and garnier to predict secondary structures The EMBOSS Nucleotide Analysis plugin includes tfscan to search for transcription factors and tcode which provides a protein coding prediction graph When these plugins are installed the protein coding prediction graph will be available under th
64. then select the 214 CHAPTER 16 WORKFLOWS Table 16 1 Add Step options for Workflows Step Purpose Function amp Add Operation Runs one of the standard Operations available in Geneious This can include other Workflows gt For Each Document For Each Sequence Extract Sequences From List P Group Documents P Group Sequences Runs the next Step in the Workflow independently on each document Runs the next Step in the Workflow independently on each sequence extracting sequences from a list if necessary Groups results from multiple operations together so that they are all used in a single invocation of the next operation Groups sequences into a sequence list document Add document chosen when running workflow Combine with Earlier Document s Save Documents Branch Prompts the user to choose a document when they first start the Workflow This document may either be chosen from anywhere in their database or from one of the documents selected when the work flow is run In the latter case the selected document will be excluded from the list of documents provided to the first Step of the Workflow If only a single document from the selected documents matches the specified document type then it will be automatically selected in stead of asking the user In both cases the selected document is com bined with each of the results from the previous Step in the Workflow See section 16 2 2 for more
65. they will not be suitable for restoring from However backups taken overnight when Geneious isn t running should be fine 4 5 BACKING UP YOUR LOCAL DOCUMENTS 59 Backing up your data directory manually is not recommended because the Geneious database structure is complex and many programs will fail to back it up properly The back up command has two options Export selected folder This will export the selected folder including all subfolders to a Geneious format file This allows you to back up an individual project within your database The backup can also be imported in to an existing database by drag and drop If you have finished working on a project it is a good idea to back it up in this way then delete it from inside Geneious to keep the size of your database down and improve the performance of Geneious You should keep archive backups in addition to these because this backup will miss your settings and data outside the selected folder Archive all data and settings This is equivalent to creating a zip archive of your entire Geneious data directory which includes all your data preferences searches and agents This option will cause Geneious to cease working on the local database while it creates the archive This type of backup cannot be directly imported in to an existing database when it is loaded everything in Geneious will revert to how it was when you took the backup Backups should be stored on another drive or can be lef
66. to select several documents geneious urnn lt URNofDocumentl gt zurn lt URNofDocument2 gt To find the URN of a document click on the small column selector button at the top right corner of the document table and enable the URN column You can then right click on the URN of a document in the table and choose Copy URN
67. to specify an optimum score such as GC content These weights are used when looking at primers whose value for this option falls below and above the optimum respectively The other weights are applied no matter in which direction they vary For details on individual options in the Primer Picking Weights dialog again hover your mouse over the option to see a short description 13 1 6 Degenerate Primer Design A degenerate primer contains a mix of bases at one or more sites They are useful when you only have the protein sequence of your gene of interest so want to allow for the degeneracy in the genetic code or when you want to isolate similar genes from a variety of species where the primer binding sites may not be identical You can design degenerate primers in Geneious by using either a sequence containing ambiguous bases or an alignment as the template and checking the Allow degeneracy box The degeneracy value that you specify is the maximum number of primers that any primer sequence is allowed to represent For example a primer which contains the nucleotide character N once and no other ambiguities has a degeneracy of 4 because N represents the four bases A C G and T A primer that contains an N and an R has degeneracy 4 x 2 8 because R represents the two bases A and G 13 1 7 Advanced Options In the Advanced panel there are options to add 5 extensions to primers and to specify a mis priming library A 5 extension can be your
68. to very low identity for the given position Figure 9 6 9 3 3 Concatenating alignments Concatenating alignments works in the same way as concatenating sequences see section 5 3 1 However for alignments to be concatenated the sequences names must be identical between alignments so that the correct sequences are matched up 9 3 4 Strip alignment columns To strip columns out of your alignment go to Tools Strip alignment columns This will create a new alignment document from your original alignment with the sites you have chosen removed Options are available for stripping columns containing all or some gaps identical sites identical sites plus transitions and ambiguous sites For the purposes of phylogenetic analyses you can also use this tool to create alignments com posed of 1st 2nd and or 3rd codon positions using the options to Strip every third codon or Strip two adjacent columns per codon 122 CHAPTER 9 SEQUENCE ALIGNMENTS Alignment View Distances Text View History Notes lt a D gt Cr Extract rc SH Translate ZY Allow Editing di Add Edit Annotatior gt gt Y Biia e gt 1 10 20 30 40 50 60 PT CITT CATGGG GAAGCAGA TTT GGGTACC ACCCAAG CCCATCAACAA Identity Te 1 Adam TTCTTTCCTAGG GAAGCAGA TTT GGGTACCTTGACTCA CCCATCAACAAC Ce 2 Harry TTCTTTCATGGG GAAGCAGA TTGGGTACC ACCCAAG CCCATCAACAA Ce 3 Sally TTCTTTCATGGGCACGAAGCAGA TTT GGGTACC ACCCAAG
69. type in a new track Expand the Properties section to enter additional properties for that annotation In the Interval section you can adjust the position of the annotation add an additional interval or mark the annotation as truncated at the 5 or 3 end 8 2 2 Importing annotations from external sources BED files GFF files and VCF files contain annotation information which can be imported into Geneious These files often do not contain the sequence itself so when you import the file you will be prompted for the reference sequence as shown in Figure 8 5 Note that if you choose use a sequence in the selected folder or you have a sequence list or more than one sequence selected and choose use a sequence in the selected documents then your sequence s name in Geneious must match the sequence name in the first column of the BED GFF or VCF file If you select a single sequence and choose use a sequence in the selected documents then your annotations will be imported onto that sequence regardless of whether it matches the sequence name in the file 102 CHAPTER 8 WORKING WITH ANNOTATIONS 08 Find sequence s Geneious can t find the sequence it needs in the selected file _ Import as a blank sequence sequence with for all its bases Use a sequence from the selected documents _ Use a sequence in the selected folder in my database _ Get the sequence from a file If Geneious still can t find the sequence i
70. until the total frequency of the topologies reaches the threshold value 160 CHAPTER 12 BUILDING PHYLOGENETIC TREES A topology threshold of 0 will result in only the most common topology being output a thresh old of 100 will result in all topologies being output 12 5 Viewing and formatting trees Once the tree is built it will appear in the Document Viewer window Figure 12 3 When viewing a tree a number of other view tabs may be available depending on the information at hand The Alignment View tab will be visible if the tree was built from a sequence alignment using Geneious The Text View shows the tree in text format Newick Tree View Alignment View Distances Text View Info ef Root Ed Swap Siblir Color Nodes T Font Save 9 aa Sea Turtle z General AAA Warbler te SS Ostrich A m c Spoonbill Zoom Moa Frog Expansion m t Tuna Ly Carp v Layout Eel Orangutan r Chimpanzee Root tengih Monkey Curvature _ PET Mangabey AAA Mouse _ Align Taxon Labels E Chipmunk gt Formatting Horse f Panda Y M Show Tip Labels Raccoon Names A A ack play Node Heights Seal Display accession oo Tiger Common Name pt Gazelle 4 Pig Dolphin Significant Digits 4 EA Max Chars 30 15 0 04 f Ser font
71. where the dotplot window sits The Colors for the Dotplot can be selected at the top of the settings panel The Classic scheme will color the dot plot lines according to the length of the match from blue for short matches to red for matches over 100 bp long For nucleotide comparisons the reverse complement can also be viewed where matches with one of the sequences reverse complemented are displayed These matches are shown by lines running from the bottom left to top right When a pairwise alignment is selected the path that the alignment takes through the dot plot can be displayed by checking Pairwise alignment path This is shown as a light blue line running through the dot plot Both of these options can 109 110 CHAPTER 9 SEQUENCE ALIGNMENTS be found under the Display section Sequence View Dotplot Virtual Gel Lengths Graph Notes gala my chimpanzee mutated a a sidad 1 000 amp 29 Jx a 1 N Colors Classic e N gt Minimap a v Display N Y Reverse complement w gt y wise alignment patr N A eh ey a f v Data Source E 4 High Sensitivity Slow v E Score Matrix Probabilistic o gt a X ln x Window Size 20 N A Threshold sog Based on the EMBOSS tool dotmatcher Tile Size 10 000 1 000 Figure 9 1 A view of dotplot of two sequences in Geneious Interpreting a Dotplot e Each axis of the plot represents a sequence e A lo
72. with splits Each branch edge in an unrooted tree corre sponds to a different split of the taxa that label the leaves of this tree 12 4 2 Creating a consensus tree of existing trees Select a tree set document e g a set of bootstrap replicate trees and choose Tree then Con sensus Tree Builder at the top of the setup dialog Check Create Consensus Tree and choose the Support Threshold you wish to use This will create a consensus tree using the trees already in the document no resampling will be performed and it will either be added to the tree document or saved as a separate tree document 12 4 3 Sort topologies This will produce one or more trees sorted by topology summarizing the results of resampling check Sort topologies under the Consensus Tree Builder options The frequency of each topol ogy in the set of original trees is calculated and the topologies are sorted by their frequency A number of these topologies based on the topology threshold will be output as summary trees The summary trees have branch lengths that are the average of the lengths of the same branch from trees with the same topology The topology threshold determines what percentage of the original tree topologies must be represented by the summarizing topologies The most common topology will always be output as the first summary tree If the frequency of this does not meet the threshold then the next most frequent topology will be added and so on
73. zoom in on the X or Y axes respectively 80 CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES Y Hide IAEA Annotations Virtual Gel Distances Text View Fasta Alignment View Info Gb Extract GERC Translate Add Edit Annotation WP Allow Editing Annotate amp Predict MM Save 9 a Consensus Identity Ce 1 Pmaj abl Ce 2 ECAl abl Ce 3 ECA2 abl Ce 4 ECA3 abl1 Ce 5 SREZ abl Ce 6 GB3 abl Ce 7 SRE3 abl Ce 8 GBl abl Ce 9 GB4 abl 108 118 128 138 148 158 167 176 VAMA oa ANON IA ste AET Jl ya W ATAAATAACCAGGTTCTCTGGCTTGGGAGHBICTTGAAAGCTGTTGGTAGRACATBTITC CTAGGAGGTGGGC dw dao lo Mba Vallada NA CTAGGAGGTGGGC TCAL ul TTT CTAGGAGGTGGGC TCAT har IWM A Mh IT aa ma W ATAAATAACCAGGTTCTCTGGCTTGGGACHICTTCGAAAGCTGTTGGTAGHACATHETC CTAGGAGGTGGGC TCAT ananaw a NN An Md wd N molaba Wi ATAAATAACCAGGTTCTCTGGCTTGGGAGMBIC TTGAAAGCTGTTGGTAGRACATBETC CTAGGAGGTGGGC TCAL Minin a aa ua wma la a NA ollo AI ATAAATAACCAGGTTCTCTGGCTTGGGAGMIC TT GAAAGCTGTTGGTAGBACATBEMT C CTAGGAGGTGGGC TCA1 Lal diva ATAAATAACCAGGTTCTCTGGCTTGCGAG TTGAAAGCTGTTGGTAGH A rc CTAGGAGGTGGCGC TCAT ae aL ac ATAAATAACCAGGTTCTCTGGCTTGGGAGMBIC T TGAAAGCTGTTGGTAGRACATBEATC CTAGGAGGTGGGC TCAL EN ho AA A PO WN luz PEN T ae Mouse over base 118 A quality 1976 residue 36 N Asn Asparagine in Consensus DE Cao AE M Show Graphs Export _ Protein
74. 0 and 100 with a higher score denoting less off target activity Selecting Score against an off target database will prompt Geneious to search sequences in the given folder for off target interactions of your CRISPR sites The sequence you are targeting can be inside this folder This way you can find CRISPR sites that target a particular gene but are scored for interactions against the entire genome The top 5 off target sites are kept and annotated onto the CRISPR site even though all off target interactions found are incorporated into the score Scoring against an off target database will significantly increase the time taken for the operation to complete Before scoring the off target database is searched for full exact matches of the whole target sequence These intervals are ignored during the scoring phase because they are likely the original target itself or an expected repeat of the target The intervals that were ignored are reported at the end of the operation in a dialog box They can be found again later by mousing over the name of the CRISPR sites annotation track in the sequence viewer If Score against an off target database is not selected the CRISPR sites will be scored against any stretches of sequence in the original document that are not part of the selected target area You can control the number of mismatches allowed between a CRISPR site and its off target sites by adjusting Maximum mismatches allowed against off targets Yo
75. 1 value This results in a value greater than 1 for over expressed genes and less than 1 for under expressed genes When one sample has no or very low expression the ratio is capped at 1 000 000 Ratio coloring reaches maximum intensity at 5 Differential Expression Log2 Ratio The base 2 log of the ratio of the normalized values between the two samples When one sample has no or very low expression the log2 ratio is capped at 1 000 000 Although results are colored by Differential Expression Confidence by default you may pre fer to switch to Differential Expression Ratio coloring once you have filtered at an appropriate confidence level e g using an abs gt 6 filter To do this click the arrow to the left of the track name and choose Color by Heatmap To export these values as a table switch to the Annotations tab above the sequence viewer then click the Track button and choose the Expression track to display Then click the Columns button and add the results columns you wish to display You can export the table in csv format by clicking Export table Chapter 12 Building Phylogenetic trees Geneious provides inbuilt algorithms for Neighbour joining Saitou amp Nei 1987 and UPGMA Mitchener amp Sokal 1957 methods of tree reconstruction which are suitable for preliminary in vestigation of relationships between newly acquired sequences For more sophisticated meth ods of phylogenetic reconstruction such as Max
76. 2 Connecting to Editing or Removing Accounts Account specific options can be accessed either from the Collaboration submenu under Tools or by right clicking on the Collaboration account in the Sources panel to bring up the account s context menu To change your account configuration including your password or email address click Edit Account Details If you change your password Geneious will attempt to change it on the 224 CHAPTER 19 COLLABORATION server the next time you connect For this purpose Geneious internally remembers your previ ous password as well so that it can still connect if you have entered your new password while disconnected To connect or disconnect from your collaboration account select Connect or Disconnect To delete your account configuration from Geneious select Delete Account Currently there is no option for deleting an account on the server 19 2 Managing Your Contacts Once you have an account and are connected you can start adding contacts You will not be able to add contacts while an account is disconnected Also you will not be able to see a contact s online status until that contact has approved your request to do so 19 2 1 Add Contact Select your account in the Sources Panel and choose Add Contact from the Collaboration sub menu or right click Ctrl click on Mac OS X on your account in the Sources Panel and choose the same option You will see a simple dialog with one field Jabber ID
77. 4747 Produ Add codon_start 1 transl_table 11 product inner membrane lipoprotein nrotain id VD NNIZRRAGZ 1 Move Dowr Edit Intervals 158 gt 4 578 3 192 399 lt 3 196 819 Move Dowr Add Edit Cancel ok Figure 8 3 The Edit Annotations Window end to mark an annotation as partial at the left end e g if a gene is incomplete at the left end when viewed on the sequence or truncated right end to denote that the feature is incomplete at the right end Bulk editing Multiple annotations can be edited at once by selecting the annotations while holding down the cntrl command key and clicking Edit Annotations Where properties contain different values in different annotations Multiple values will be shown next to that property Editing a property will change the value to be the same for all annotations Intervals cannot be edited when multiple annotations are selected 100 CHAPTER 8 WORKING WITH ANNOTATIONS 8 1 4 Extracting Annotations To extract an annotation to a separate document select it either on the sequence or in the An notations Table and click Extract If you want to actively link the extracted annotation to the source document so that changes on the source document are propagated to the extracted document check Actively link source and extracted documents If the annotation you are extracting contains multiple intervals the intervals can be concate nated into a single sequence If
78. 50 125bp Y AMA GFP3 x GFP4 x GFPS amp GFP6 x Primer amp Enzyme CAAT GTTA PCR Product TTTA ARA PCR Product TGTA ACAT Enzyme amp Primer ACCT 251 375 125bp 19 139 121bp Y 153bp Y 76 143 68bp Y Auto Arrange _ Choose Insert M Save used primers Save intermediate products M Save in sub folder Golden Gate a Cancel OK Figure 14 5 Golden Gate setup dialog showing backbone and inserts with a range of different reaction types The Backbone is the sequence e g a vector into which the inserts are ligated This setting will always remember the previously used backbone and can be manually changed by either using the dropdown showing recently used backbones and any of the currently selected se quences or selecting a backbone with the Choose button If a backbone is set the product will always be circular and the Circular Product option will be disabled If None is chosen as the backbone the product can be circularized by checking Circular Product The Insert s can either be selected in the document table prior to opening the Golden Gate options or can be added from within the setup options by clicking the Choose Insert button Inserts can also be removed by clicking the small X in the top right corner of the insert tag Each insert has a small dropdown arrow by which the sequence can be reverse complemented The Enzyme can be chosen from any of the commercially available Type IIS enzymes that have
79. 50 000 366 924 P 7 ChM 3 Y zE e e 100 2 laiz a E Annotations and Tracks a as SS i Mm Show Annotations 4 460 of 4 484 Al Q El M cps 560 gt y lt gt Hall M Exon 656 y lt gt lo 0 21 170 21 180 21 190 Five_prime_UTR 8 PP y lt gt gt ChrM 3AGACGATGGAATGCTATGGGATGGATGG TAGAG C M Gene 438 gt y lt gt v Microsatellites e M mRNA 536 lt gt v Known Genes HARE a y Redundant G m rRNA 3 of 6 AS pa Prediced ORF M tRNA 21 of 42 lt gt chromosome TAIR9 Mt 1 366924 1 Tracks Opti y intron 555031 j a z intron 11 M Microsatellites 647 lt gt NS TAIR10_gene a M Prediced ORF 1 554 y lt gt ATMGODO60 TAIR G ATMGO0060 1 Mi Redundant Genes 146 y lt gt ATMGO0060 1 TAIR M Known Genes 430 Options lt gt ATMGO0060 1 TAIR M CDs 140 y 4 gt M Exon 164 o y lt gt M Five_prime_UTR 4 v lt gt M mRNA 122 o y lt gt C microRNA 24 Options lt gt on lt gt f rRNA 3 Gee lt gt y Figure 8 2 The annotations options in the sequence viewer Individual annotation types can be turned on or off using the checkboxes to the left of the annotation type or all annotations can be turned off by unchecking Show Annotations at the top of the panel Note that turning an annotation type off does not remove it from the sequence it only hides it from view Directly benea
80. 80 Consensus ACGGTACCTG CAGAAGAAG CA CCGG CCAACTACG TGC CAGCAG CCC gion 2 Coverage io AE 490 GCGG Ce REV 1 Fragmen AC Ce FWD 2 Fragmen D REV 3 Fragmen Figure 10 1 Click and drag the trims to adjust If you choose to trim your sequences at the assembly step the sequences are trimmed and as sembled in one operation and you will not be able to view the trimming that Geneious uses before assembly is performed However the trimmed regions will still be available and ad justable after assembly is complete If you choose to trim your sequences prior to assembly select Use Existing Trim Regions when you set up the assembly Trimmed annotations can also be created manually using the annotation editing in the sequence viewer If you create annotations of type trimmed and save them then Geneious will treat them the same as ones generated automatically and they will be ignored during assembly Trimmed annotations can also be modified in this way before or after assembly Trim Ends options Annotate new trimmed regions Calculate new trimmed regions and annotate them the trimmed regions will be ignored when performing assembly and calculating the consen sus sequence 10 1 READ PROCESSING 127 Annotate new trimmed regions ignored by assembly and consensus Remove new trimmed regions from sequences Remove existing trimmed regions from sequences trim vectors UniVec High sen
81. A it will be labelled RNA Fold Figure 7 1 The fold prediction is performed by the Vienna package RNAfold tool Information on the options for this tool can be found at the following web page http www tbi univie ac at ivo RNA RNAfold html The View Options allow you to turn off on and color the bases flip the coordinates highlight the start blue and end red of the sequence and rotate the model As with other viewers you can zoom in on the model and drag the view around or use the scrollwheel using the same keyboard modifiers as the sequence viewer Selection is synchronized between the sequence view and the fold view In addition when in split view mode the fold viewer will scroll to the selected area when zoomed in By default Color by probability is used where red bases are the ones with the strongest proba bility of the bases being paired with each other in paired regions or being unpaired in unpaired regions Green is the middle ground and blue is the lowest probability Color by probability is only available when using the Partition Function Compute Options will rerun RNAfold when you change their settings so depending on the size of the sequence there may be a noticeable recompute time 91 92 CHAPTER 7 RNA DNA AND PROTEIN STRUCTURE VIEWER Sequence View Dotplot Self Annotations RNA Fold Text View History Notes Hao 2 P 138 2 NES View Options Color By Probability 15 Mm Show Bases Mm Sh
82. ACGCCGGAACCGACACCTGATCCTGAGCCAACAC mum re a A dad a A iv A son 8866 a a son dad mum A Figure 8 7 Example 2 Compare annotations setup above and results below for finding polymorphisms in a child which are not present in either parent Chapter 9 Sequence alignments 9 1 Dotplots A dotplot compares two sequences against each other and helps identify similar regions Using this tool it can be determined whether a similarity between the two sequences is global present from start to end or local present in patches To view a dotplot in Geneious select two nucleotide or protein sequences in the Document Table and select Dotplot in the tab above the sequence viewer Figure 9 1 If a single nucleotide or protein sequence is selected then a dot plot showing a comparison of the sequence to itself can be viewed The Geneious dotplot offers two different comparison engines based on the EMBOSS dottup and dotmatcher programs You can choose which program to use by setting the sensitivity under Data Source the panel to the right of the dot plot The Low Sensitivity Fast setting uses dottup and the High Sensitivity Slow setting uses dotmat cher More information on these programs can be found by going to http emboss sourceforge net The dotplot is drawn from top left to bottom right The Minimap in the panel to the right of the viewer aids navigation of large dotplots by showing the overall comparison and a box indicating
83. AUP Yes Yes Geneious Assembler Mapper Yes Yes Bowtie short read mapper Yes Yes BBMap short read mapper Yes Yes BWA short read mapper No Yes Mag short read mapper No Yes SOAP2 short read mapper No Yes Tophat RNAseq aligner Yes Yes Velvet short read assembler Yes Yes Tadpole de novo assembler Yes Yes Merge Paired Reads using BBMerge Yes Yes Remove Duplicate Reads using BBTools Yes Yes Trim using BBDuk Yes Yes Find Variations SNPs with SAMtools No Yes CustomBLAST Yes Yes 238 CHAPTER 21 GENEIOUS SERVER Chapter 22 Advanced Administration 22 1 Default data location By default the data location will be in the user s home directory You can change this by setting an environment variable which will be used by the Geneious launcher such as setting a HOME variable to be where you want a user to store their data On Windows and Linux edit the Geneious in use vmoptions file in the installation di rectory and add DdataDirectoryRoot HOMES Geneious on a new line after the other settings On Mac OS X edit the Applications Geneious app Contents Info plist and find the lt key gt Argument s lt key gt section to match the following lt key gt Arguments lt key gt lt string gt distributionVersion DdataDirectoryRoot SHOMES Geneious lt string gt A special JAVA_USER_HOMES variable is normally used which resolves to user home and is what Geneious uses by default The program will create a Geneio
84. Available for chromatogram documents which have quality confidence values The ends are trimmed using the modified Mott algorithm see be low based on these quality values Richard Mott personal communication e Maximum low quality bases Specifies the maximum number of low quality bases that can be in the untrimmed region Low Quality is normally defined as confidence of 20 or less This can be adjusted on the Sequencing and Assembly tab of Preferences e Maximum Ambiguities Finds the longest region in the sequence with no more N s than the maximum ambiguous bases value and trims what is not in this region This should be used when sequences have no quality information attached e Trim 5 End and Trim 3 End These can be set to specify trimming of only the 3 or 5 end of the sequence A minimum amount that must be trimmed from each end can also be specified e Maximum length after trim If the untrimmed region is longer than the specified limit then the remainder will be trimmed from the 3 end of the sequence until it is this length The Modified Mott algorithm The modified Mott algorithm for trimming ends based on quality operates as follows For each base it subtracts the base error probability from an error probability cutoff value default 0 05 to form the base score The base error probability is calculated from the quality score Q such that P error 10 9 10 This means that low quality bases have high error pr
85. CATTACCACCA Translatior EKO RENS T T HET T _ as A Nucleotides 2 ae C Complement 50 60 70 80 230 240 250 260 Translation CCAT CACCAT TACCACAGGT AACGGT GCGGGCT GAC GCGT Translation Options T T SGN EGE A CGE p Frame By selection or annotation slat Genetic Code Bacterial E coli K12 MG1655 Translatior 90 100 110 120 j 300 Colors ARND Edit 270 280 290 E coli K12 MG1655 ACAGGAAACACAGAAAAAAGCCCGCACCTGACAGTGCGGG a zs ojo o oe ae 130 140 150 Translatior 310 320 330 E coli K12 MG165S ada i Cursor before base 151 original base 331 Figure 5 5 Translating a CDS 5 5 1 Genetic Codes Geneious supports a range of genetic codes which can be chosen from the drop down menu in the Translation options To set a default genetic code which will apply to all documents in your database click the Settings cog next to the drop down menu You can also enter a custom genetic code here by clicking Add and editing an existing genetic code template 5 5 2 Back Translating To create a nucleotide sequence from a protein document go to Sequence Back Translate Ambiguous back translation uses a specific genetic code to produce a nucleotide sequence with ambiguous bases so that every possible codon is represented for each amino acid Un ambiguous back translation uses codon usage tables for selected organisms to produce a nu cleotide sequence where
86. Coding Prediction solls Window Size 200 Step Size Ey Based on the EMBOSS 6 5 7 tool tcode C GC Content 100 Show AT Graph Frame plot Sliding Window Size 48 Y Chromatogram 8 Ma Mc Mc MT Moua C Coverage 3015 Y Log Scale Highlight below oje V Highlight above soji Highlight single strand MW W Minimum MW Mean Maximu L Sequence Logo 50 Weight by quality 40 Figure 5 6 A sequence alignment containing chromatograms with quality scores enabled 5 7 META DATA 81 5 6 1 Binning by quality Chromatograms can be binned on the basis of their quality scores into Low Medium or High quality bins The parameters for each of these bins are set under Tools Preferences Se quencing see section 1 2 1 for more information To see the bin for a trace add the Bin column to the document table by going to View Table Columns You can also view the percentage of bases that are low medium or high quality by adding LO MO and HQ columns to the document table 5 7 Meta data Meta data is additional information you can add to any of your local documents for example sample collection history organism identification primers used etc Meta data is added in the Properties view under the Info tab This tab displays standard properties of documents such as the name and description plus any meta data you have added Any meta data that you add can appear in
87. Data Type to add or remove a field from the type click the or buttons to the right of the field Field type This describes the kind of information that the column contains such as Text Integer and True False The full list of choices in Geneious is shown in figure 5 9 Constraints These are limiting factors on the data and are specific to each field type For ex ample numbers have numerical constraints is greater than is less than is greater or equal to and is less or equal to These can be changed to suit The constraints for each field can be viewed by clicking the View Constraints button next to the field This will show a pop up menu with the constraints you have chosen see figure 5 8 000 Constraints for is greater than t is less than is greater or equal is less or equal Clear Constraints Cancel Figure 5 8 The Edit Constraints window 5 7 4 Editing Meta Data Types To edit meta data types e g by adding and removing fields click Edit Meta Data Types Select the meta data type you want to edit and then add remove or edit the fields as described in section 5 7 3 5 7 5 Using Meta Data The main purpose of meta data is to add user defined information to Geneious documents However meta data can be searched for and filtered as well Also documents can be sorted according to meta data values Searching Once meta data is added to a document it is automatically added to t
88. Description x Primer Extension Tag column 3 X Additional Fields Organism X Common Name y Taxonomy X Topology m Genetic Code X Molecule Type Accession X Created X Notos Note Type X de Reset to Defaults Cancel _ Reset to Defaults _ Cancel Figure 13 5 Importing primers from a spreadsheet You will be asked which type of sequence you are importing When you choose to import primers or probes you will receive some options that allow you to determine characteristics for them as an extra step Immediately below this is a preview of the first few rows of data and a checkbox that allows you to tell Geneious that the top row is a heading row and should be ignored Below the preview is a list of common and additional fields along with dropdown boxes These boxes allow you to specify which column contains which piece of data often one or more of these won t be applicable and can be left as None Note that at minimum you must specify a Sequence field 174 CHAPTER 13 PCR PRIMERS Lastly any add additional data in the form of meta data Clicking the dropdown box next to Meta Data at the bottom of the dialog will allow you to import values to meta data and clicking the or will allow you to insert or remove additional meta data types Next click the Fields button to bring up a dialog An additional set of dropdown boxes will allow you to specify again which columns of data contain the fields w
89. Genbank document should be copied to the Name field in Geneious Sequencing This tab has options for the management of trace files and assemblies allowing you to set thresholds that assign sequences as low medium or high quality To change the default pa rameters or set up a new binning profile click the Default profile then click Edit View The following options are available e Confidence Set the threshold values of base call confidence used to determine if a base call is low medium or high quality This affects the binning parameters described below as well as the Base Call Quality color scheme in the Sequence View e Sequence binning options Specifies the requirements for individual traces to be binned as medium or high quality overall To see the Bin for a trace turn on the Bin column under Table Columns in the View menu Assembly binning options Specifies the requirements for assembly documents to be binned as medium or high quality overall To see the Bin for an assembly turn on the Bin column under Table Columns in the View menu To create a new profile change the parameters how you wish then enter a new Profile name and click OK Other options for managing quality bins are e Track binning history in meta data When turned on meta data will be added to traces when they are trimmed see the Properties view tab This meta data will then be updated every time the trace is re trimmed maintaining a history of the tr
90. LASTZ plugin file Install this file into your copy of Geneious to install LASTZ plugin Download the Mafft plugin file Install this file into your copy of Geneious to install Mafft plugin Download the Maq plugin file Install this file into your copy of Geneious to install Maq plugin Download the Mauve plugin file Install this file into your copy of Geneious to install Mauve plugin Download the MrBayes plugin file Admin Console Click to access administrative console if you are a server administrator Figure 21 1 Download Geneious Server Plugins Click on each plugin to download it and once you ve downloaded all plugins drag them from your downloads folder into Geneious You ll probably have to restart Geneious after all plugins have been installed Note that it may take some time for the plugins to install so give it some time Once it is clear the plugins have all installed restart and when Geneious comes back up you should now see the Geneious Server link in the Sources Panel Click this and you ll see a button to log in Use the log in button to display a dialogue requiring the hostname username and password details which your administrator should have provided you with Figure 21 2 Once you ve logged into the server you will now have access to the shared database space which will appear under Shared Databases in the Sources panel We recommend you create a folder for your own documents The benefits of this folder is that th
91. ND EXPORTING DATA Newick tree The Newick format is commonly used to represent phylogenetic trees such as those inferred from multiple sequence alignments Newick trees use pairs of parentheses to group related taxa separated by a comma Some trees include numbers branch lengths that indicate the distance on the evolutionary tree from that taxa to its most recent ancestor If these branch lengths are present they are prefixed with a colon The Newick format is produced by phy logeny programs such as PHYLIP PAUP Tree Puzzle and PHYML Geneious can import and export trees including bootstrap values and branch lengths in Newick format Nexus tree The Nexus format was designed to standardize the exchange of phylogenetic data including sequences trees distance matrices and so on The format is composed of a number of blocks such as TAXA TREES and CHARACTERS Each block contains pre defined fields Geneious imports and exports files in Nexus format and can process the information stored in them for analysis If you want to export a tree in a format that preserves bootstrap values make sure you export with metacomments enabled otherwise the bootstraps will be lost PDB structure Protein Databank files contain a list of XYZ co ordinates that describe the position of atoms in a protein These are then used to generate a 3D model which is usually viewed with Rasmol or SPDB viewer Geneious can read PDB format files and display a
92. S site Geneious will assume you wish to use this and will only design a second opposite orientation primer based on the appropriate rule for PCR 4 Existing primer_bind annotations with other extensions If Geneious detects a primer bind annotation with an extension which does not contain a valid type IIS site then the 5 ter minus of the extension will be considered the fusion point and the extension will be fur ther extended to introduce a valid type IIS recognition site resulting in a new primer sequence 5 Existing primer_bind annotation s without extension s If a primer_bind annotation without an extension is found then an extension will be appended to introduce a valid type IIS recognition site resulting in a new primer sequence 6 Blunt ends If Geneious finds a blunt end and no suitable type IIS sites or primer_bind annotations are present then a primer with an appropriate type IIS site extension will be designed The fusion point will be the termini us of the blunt end fragment For any of these Geneious only accepts cut sites where the recognition site will be cut out or primers that point towards each other Important 14 7 TOPO CLONING 195 e Removal of unwanted internal Type IIS sites If one or more of your sequences contain the specified type IIS restriction site s then Geneious will assume you want to use the site s in the assembly process and design a strategy accordingly If one or more of your seq
93. Star seq pro Nucleotide amp protein sequences DNAStar DNA Strider str Sequences DNA Strider Mac program ApE Embl UniProt embl swp Sequences Embl UniProt Endnote 8 0 or 9 0 XML xml Journal article references Endnote Journal article websites FASTA fasta fas fasta gz etc Sequences alignments PAUP ClustalX BLAST FASTA FASTQ fastq fq fastq gz etc Sequences with quality Illumina and other NGS sequencers GCG seq Sequences GCG GenBank gb xml Nucleotide amp protein sequences GenBank Geneious xml geneious Preferences databases Geneious Geneious Education tutorial zip Tutorial assignment etc Geneious GFF gff Annotations Sanger Artemis MEGA meg Alignments MEGA Molecular structure pdb mol xyz cml gpr hin nwo 3D molecular structures 3D structure databases and programs Newick tre tree etc Phylogenetic trees PHYLIP Tree Puzzle PAUP ClustalX Nexus nxs nex Trees Alignments PAUP Mesquite MrBayes amp MacClade PDB pdb 3D Protein structures SP3 SP2 SPARKS Protein Data Bank PDF pdf Documents presentations Adobe Writer IATEX Miktex Phrap ACE ace Contig assemblies Phrap Consed PileUp msf Alignments pileup gcg PIR NBRF pir Sequences alignments NBRF PIR Qual qual Quality file Associated with a FASTA file Raw sequence text seq Sequences Any file that contains only a sequence Rich Sequence Format rsf Sequences alignments GCGs NetFetch Comma Tab Sepa
94. UM and PAM matrices are substitution matrices The number of a BLOSUM matrix indicates the threshold similarity between the sequences originally used to create the O Dayhoff ed Atlas of protein sequence and structure vol 5 National biomedical research foundation Washington DC 1978 112 CHAPTER 9 SEQUENCE ALIGNMENTS matrix BLOSUM matrices with higher numbers are more suitable for aligning closely related sequences For PAM the lower numbered tables are for closely related sequences and higher numbered PAMs are for more distant groups When aligning protein sequences in Geneious a number of BLOSUM and PAM matrices are available Algorithms for pairwise alignments Once a scoring system has been chosen we need an algorithm to find the optimal alignment of two sequences This is done by inserting gaps in order to maximize the alignment score If the sequences are related along their entire sequence a global alignment is appropriate However if the relatedness of the sequences is unknown or they are expected to share only small regions of similarity such as a common domain then a local alignment is more appropriate An efficient algorithm for global alignment was described by Needleman and Wunsch 1970 and their algorithms was later extended by Gotoh 1982 to model gaps more accurately For local alignments the Smith Waterman algorithm is the most commonly used See the references at the links provided for further informa
95. Vectors and or other inserts can then be chosen from within the Gibson Assembly setup window by clicking the Choose button This brings up a document chooser from which you can select your vector and or inserts from any folder in your database e If none is selected as the Vector the product s will be linear otherwise circular 190 CHAPTER 14 CLONING e Insert sequences can be ordered via drag n drop Sequences that have been previously grouped into lists e g Promoters shown in brown ish will all be inserted at the specified position generating one product per sequence at that position e If no grouped documents are provided in the drag n drop field the user can chose to insert the sequences either all at once into the vector as a sequential assembly Assemble fragments end on end or alternatively as separate inserts with one insert per generated product Insert each fragment separately e The minimum overlap length of the complementary sequence can be specified as well as the minimum melting temperature of the complementary sequence Min Overlap Tm e To calculate the Tm a collapsible field is available showing the options provided and required by primer3 The operation will remove 5 overhangs and fill up 3 overhangs that are eventually present on the sequences eg when they derived from a restriction digestion The possible different se quence combinations are created and complementary extremities that might be alre
96. _ LEFT RIGHT SEQUENCE Product Size Product Size Ranges PRIMER PRODUCT SIZE Pair Hairpin PAIR ANY COMPL PRIMER _ PAIR COMPL_ANY Pair Primer Dimer PAIR 3 COMPL PRIMER PAIR COMPL_END Pair Tm Diff Max Tm Difference PRIMER _PRODUCT_TM_OLIGO_TM_DIFF Table 13 1 Geneious primer characteristics and their Primer3 counterparts 13 1 9 Output from Primer Design Once the task and options have been set click the OK button to design the primers A progress bar may appear for a short time while the process completes When complete primers and probes will be added as annotations on the sequences The annotations will be labelled with the base number the primer starts at followed by either F forward primer R reverse primer or P probe Primers will be coloured green and probes red Detailed information such as melting point tendency to form primer dimers and GC content can be seen by hovering the mouse over the primer annotation The information will be pre sented in a popup box Alternatively double clicking on an annotation will display its details in the annotation editing dialog Table 13 1 shows how the values in the Geneious primer an notation map to the original Primer3 values If you are designing primers off an alignment the primer will be designed on the consensus sequence by default To design primers for every sequence in the alignment and have the primers annotated separately on each sequence
97. ad the reference sequence can be chosen from within the Map to Refer ence setup options by clicking Choose This brings up a document chooser from which you can select single or multiple reference sequences from any folder in your database Multiple reference sequences may be selected either by choosing a sequence list containing your reference sequences or by selecting multiple sequence documents using the Choose button Single reference sequences may be pre selected prior to choosing the Map to reference option however when using the Choose button only those documents selected within the Choose dialog will be used as reference sequences all other sequences will be mapped If multiple reference sequences are selected each read will be mapped to the sequence with the best match only and will produce one contig per reference Batch assemblies where each read 10 3 MAP TO REFERENCE 137 gets mapped to each reference sequence can be done by using Workflows Map reads to each reference sequence 10 3 2 Fine tuning When aligning to reference the sequences are not aligned to each other each of them is instead aligned to the reference sequence independently and the pairwise alignments are combined into a contig However an iterative fine tuning step can be enabled which makes reads that overlap from the initial assembly stage align better to each other Fine tuning causes reads to align better to each other around indels whic
98. ads must support the discovery of a junction in order for it to be annotated This threshold can be adjusted under More Options by changing the Minimum support for intron fusion gene discovery setting If Span annotated mRNA introns is checked junctions will be created from existing annota tions on the reference sequence Reads are still allowed to map anywhere but will be allowed to freely span these junctions if that produces the best mapping 10 3 MAP TO REFERENCE 139 To only find introns up to a certain size check Find novel introns up to to find introns of any size or structural rearrangements that may indicate a fusion gene use Find fusion genes and novel introns As for deletion and structural variant discovery junctions are annotated on the reference se quence under a track named after the reads Each junction has the following properties Junction Type For introns under 2 000 000 bp this will be Intron For longer introns or structural variants this will be Fusion with inversion potentially appended Intervals Each junction consists of two annotation intervals of a single nucleotide in length which cover the final nucleotide used in the reference sequence before the read jumps and continues elsewhere For junctions of type Intron the junction is represented as a single annotation with two linked intervals For introns that have common start and finish nucleotides the intervals will be assigned an appropriate dire
99. ady present will be considered when primers get designed For sequences without complementary overlaps a pair of primers will be generated If both or only one of the ends are complementary primers for both ends will be created since the sequence will still have to be modified by a Primer Extension PCR to make it compatible at the opposing end If both are complementary no primers will be generated Extensions will be added to the primer corresponding to the neighboring sequence Modifica tions that have been manually introduced at the extremities of a vector will be automatically added as part of the extension so that they get introduced to the sequence during the PCR Manual modifications to the ends of insert sequences should be annotated as type Gibson Primer Extension in order to be added to the primer extension rather than the binding se quence The melting temperature is calculated for the primer binding sequence and the extension part including the modified bases For both primer and extension generation the user specified Tm formula is used In many cases the Phusion DNA polymerase is used for which it is recom mended to use the Tm formula of Breslauer et al 1986 Primers are generated only for insert sequences supposing that the vector should stay unmod ified For this reason the extension length of the primer extending to the vector will be twice as long the full specified minimal overlap length compared to extensions on
100. ain toolbar and the document table look and also show or hide tips and the memory usage bar In the Behavior panel you can change the way newly created documents are handled such as where they should be saved to and whether they are selected straight away You can choose whether to store document history and create active parent descendant links see section 6 Reset questions allows you to reset the questions where you have previously told Geneious to remember your preference If you have checked remember my preference in a dialog window in Geneious that window will no longer appear You can click the Reset questions button to get these windows to appear again In the Viewer panel you can set whether the same view settings are saved across documents of the same type Keyboard This section contains a list of Geneious functions and allows you to define keyboard shortcuts for them Shortcuts that are already defined are highlighted in blue Setting shortcuts can 12 CHAPTER 1 GETTING STARTED help you quickly navigate through Geneious without using the mouse and also allows you to redefine shortcuts to ones you may be familiar with from other programs Double click on a function to bring up a window to enter your new keyboard shortcut If you use one that is already assigned Geneious will tell you what function currently has that shortcut NCBI Here you can set the URL for the NCBI BLAST database and specify which field of the
101. aining only the sequence span ning and including the PCR primers Any 5 extensions on the primers will also be included 13 10 More Information The Primer feature in Geneious is based on the program Primer3 http bioinfo ut ee primer3 Copyright c 1996 1997 1998 1999 2000 2001 2004 Whitehead Institute for Biomedical Research All rights reserved If you use the primer design feature of Geneious for publication we request that you cite Primer3 as Steve Rozen and Helen J Skaletsky 2000 Primer3 on the WWW for general users and for biologist programmers In Krawetz S Misener S eds Bioinformatics Methods and Protocols Methods in Molecular Biology Humana Press Totowa NJ pp 365 386 Source code available athttp sourceforge net projects primer3 Further information on the functionality of the primer design feature can be found in the primer3 documentation available here http primer3 ut ee primer3web_help htm Please note that some controls have been changed renamed or removed from Geneious but most of the primer3 functionality is available 180 CHAPTER 13 PCR PRIMERS Chapter 14 Cloning The cloning features in Geneious allow you to simulate several different types of cloning in cluding basic restriction cloning Topo cloning Gateway cloning and Gibson Assembly You can also find restriction sites on your sequence of interest and simulate digestion and ligation reactions The following options are availabl
102. alW Alignment Consensus Align Profile Align a Mauve Genome Cost Matrix 65 similarity 5 0 4 0 B Gap open penalty B Gap extension penalty 3 8 Alignment type Global alignment with free end gaps B _ Automatically determine sequences direction PF Build guide tree via alignment faster Refinement Options 5 A E C Refinement iterations 215 Restore Defaults Cancel C Figure 9 3 The multiple alignment window You can also do a multiple alignment via translation and back as with pairwise alignment see section 9 2 6 116 CHAPTER 9 SEQUENCE ALIGNMENTS 9 2 3 Sequence alignment using ClustalW ClustalW is a widely used program for performing sequence alignment Geneious allows you to run ClustalW directly from inside the program without having to export or import your sequences To perform an alignment using ClustalW select the sequences or alignment you wish to align then select Align Assemble Multiple Align Select ClustalW as the alignment type and the options available for a ClustalW alignment will be displayed The options are e Cost Matrix Use this to select the desired cost matrix for the alignment The available options here will change according to the type of the sequences you wish to align You can also click the Custom File button to use a cost matrix that you have on your computer the format of these is the same as for the program BLAST e Gap open cost and Gap extend
103. all documents with AttB AttP sites followed by an LR reaction on the results of the BP reaction and any input documents with AttL AttR sites For example to insert a PCR product with attB sites directly into a destination vector select the PCR product a donor vector and a destination vector Geneious will first produce an entry clone from the PCR product and donor vector then react this entry clone with the destination vector to produce an expression clone Annotate att sites This operation searches for AttB AttP AttL and AttR sites and annotates them on your se quence Add AttB Sites to PCR product This operation allows you to add AttB sites to a PCR product It will work on the following types of document e A PCR product AttB sites will be appended to the PCR product e A document with primer binding sites annotated If there is more than one pair Geneious will ask you which pair to use The PCR product will be extracted and AttB sites ap pended 192 CHAPTER 14 CLONING 14 6 Golden Gate Golden Gate is a method to conveniently digest and ligate multiple sequences with the same Type IIS enzyme in a single reaction The setup dialog for Golden Gate assembly is shown in Figure 14 5 e0 Golden Gate Backbone pGoldenGate SE7 s Choose Enzyme Bsal gt Y Circular Product Backbone GFP1 3 GFP2 z TGGA Cut by Bsal TCAT AGTA Full length PCR AT TACC Cut by Bsal 3 364 2 678 6 566bp 125bp vV ATGG 126 2
104. allows you to set which part of the sequence to extract e g just the annotated region or the entire sequence based on the criteria you have set To extract regions of sequence upstream or downstream of the annotated sequence enter the length of the additional sequence you want to extract under Extraction Context Intersecting Annotations allows you to set what to do with other annotations that don t match the criteria but which overlap with the matched region If there are multiple annotations on one sequence which match the criteria these can be concatenated into one sequence by checking Concatenate regions within each sequence 8 2 Adding annotations Geneious has many functions for adding annotations to sequences They can be added man ually imported from external sources transferred from other sequences or added as part of structure or gene prediction steps Each of these options is described in the sections below For more information on viewing and editing annotations see sections 8 and 8 1 2 8 2 1 Manual creation of annotations To create an annotation on your sequence or alignment select the region of the sequence where you wish to place the annotation and click the Add Annotation button In the Add Annotation dialog enter an annotation name and select a existing type or type a new one If you wish to put the annotation on a track rather than directly on the sequence either choose an existing track from the drop down menu or
105. ally update the primer sequence and characteristics A 5 extension can also be added directly onto a primer in this step by clicking the button next to Extension See section 13 8 for more information on adding 5 extensions 800 Add annotation Type primer_bind L Track No Track Direction e Forward _ Reverse Undirected Binding site 54 to 71 3 Primer Sequence CCATGGCCCTGTGGATGC Extension none Characteristics Length 18 Hairpin Tm None Tm 60 8 Self Dimer Tm 12 3 GC 66 7 gt Properties Cancel ok Sc Figure 13 4 Create a primer by adding a primer annotation 13 3 Importing primers from a spreadsheet You can import primers and probes directly into Geneious from Comma Tab Separated Values documents You can either import them from the Import From File menu or simply paste 13 3 IMPORTING PRIMERS FROM A SPREADSHEET the contents of the document into Geneious 173 When Geneious has successfully recognized the file as CSV or TSV you will see the following dialogue Figure 13 5 amp Import Sequences ES Import Type Primer v Determine Characteristics Options v Top row values are column headings Primers Tag No Tag Primer Tag Primer Fish_1651F 1 AACCGA GACGAKAA AACCGAGA Fish_1651F 2 TAGAGC GACGAKAA TAGAGCGA Fish_16S1F 3 GAAGAG GACGAKAA GAAGAGGA lt nal Name Primers column 1 X Sequence Primer column 4 X
106. an coalescent model is used to describe the time structure of the ancestral tree We recover information about the unknown true ancestral coalescent tree population size and the overall mutation rate from temporally spaced data that is from nucleotide sequences gathered at different times from different individuals in an evolving haploid population We briefly discuss the methodological implications and show what can be inferred in various practically relevant states of prior knowledge We develop extensions for exponentially growing population size and joint estimation of substitution model parameters We illustrate some of the important features of this approach on a genealogy of HIV 1 envelope env partial sequences PMID 12136032 Figure 3 2 Viewing bibliographic information in Geneious 3 4 Agents Databases searches can be automated using an Agent allowing you to continuously receive the latest information on genomes sequences and protein structures Each agent is a user defined automated search You can instruct an agent to search any Geneious accessible database at reg ular intervals e g weekly including your contacts on Collaboration This simple but powerful feature ensures that you never miss that critical article or DNA sequence To manage agents click on the agent icon in the toolbar An agent has to be set up before it can be used 3 4 AGENTS 45 3 4 1 Creating agents To set up an Agent click the Agents icon
107. and the Create button You now need to specify a set of search criteria including the database to search key words to search on search frequency and the folder you wish the agent to deliver its results to The search frequency may be specified in minutes hours days or weeks You can only use whole numbers Selecting Only get documents created after today will cause the agent to check what docu ments are currently available when the agent is created Then when the agent searches it will only get documents that are new since it was created This is useful if for example you have already read all publications by a particular author and you want the agent to only get new publications Alternatively you can click the Create Agent button which is available in some advanced search panels This will use the advanced search options you have entered to create the agent The easiest way to organize your search results is to create a new folder and name it appro priately You can do that by navigating to the parent folder in the Deliver to box and clicking New Folder or by creating a new folder beforehand as follows 1 Right click Ctrl click on Mac OS X on the Sample Documents or Local folders This brings up a popup menu with a New Folder option 2 Create a new folder and name it according to the contents of the search For example type CytB if searching for cytochrome b complex 3 Once created select the new folder You can
108. ange the name type or properties of a given annotation type or move the annotation to or from a track This applies the change to all annotations of this type e Delete all type annotations Deletes all annotations of a particular type The popup menu for individual tracks has an additional option Color by Heatmap This will color annotations on that track according to the contents of a qualifier field enabling the creation of annotation heatmaps by using a score value or some other metric stored in the qualifier of an annotation The way annotations are drawn on the sequence can be further customized in the Advanced tab of the sequence viewer see section 5 2 8 8 1 2 The Annotations Table The Annotations tab appears above the sequence viewer whenever sequences containing an notations are selected It displays each annotation as a row in a table with columns corre sponding to the qualifiers for the annotations Selection of annotations is synchronised with other viewers such as the sequence viewer and dotplot To change what is displayed in the annotations table use the buttons above the table 98 CHAPTER 8 WORKING WITH ANNOTATIONS e Types allows you to specify what annotation types are displayed in the table The Select One button in the menu is a quick way to view just one type while also select ing the relevant columns for that type Relevant columns are deemed to be ones where at least one annotation of that type has
109. ants of other documents each with their own parents and so on In many situations it is useful to preserve this hierarchy so that future alterations for example the re calling of a base or the addition of a new annotation can be transferred downstream to the molecules affected by this change in a parent An active link between a child and its parents means that when you modify any of the parent documents you will be given the choice of propagating these changes to the child When this modification affects a part of the parent involved in creating the child the change will be immediately visible in the child Modifications include things like editing the residues of a sequence adding new annotations or changing the meta data associated with the document Propagating a change to a parent document causes Geneious to rerun every operation that links that parent actively to one or more child documents with the altered parent document and any other parents as input Geneious stores the options that the operation was originally run with so that it can reproduce the original operation s conditions exactly and afterwards matches up the newly regenerated child documents with any former children and replace their contents where possible Occasionally one or more of the parent documents has been altered to a point where an op eration can no longer be rerun or a necessary parent document is inaccessible In this case Geneious will inform you of th
110. as an outgroup or leave it as no outgroup to build an unrooted tree 12 2 TREE BUILDING IN GENEIOUS 155 42 Geneious Tree Builder Consensus Tree Builder RAxML jp MrBayes Garli PHYML FastTree Genetic Distance Model Tree build Method Neighbor Joining Outgroup No Outgroup Pairwise distances will be obtained from the multiple sequence alignment This may reduce accuracy slightly but will produce results faster Consensus Tree Options C Resample tree Resampling Method Bootstrap Random Seed 7 411 8 Number of replicates 100 o Create Consensus Tree Sort Topologies Support Threshold 50 8 Topology Threshold ofl _ Save raw trees Figure 12 2 Tree building options in Geneious 156 CHAPTER 12 BUILDING PHYLOGENETIC TREES e Resample tree Check this to perform resampling e Resampling method Either bootstrapping or jackknifing can be performed when resam pling columns of the sequence alignment e Number of samples The number of alignments and trees to generate while resampling A value of at least 100 is recommended e Create Consensus Tree Choose this to create a consensus tree from the resampled data Sort Topologies Produce trees which summarise the topologies resulting from resam pling Support threshold This is used to decide which monophyletic clades to include in the consensus tree after comparing all the trees in the original set
111. ate A 50 jackknife randomly deletes half of the columns from the alignment to create each pseudo replicate 12 4 RESAMPLING BOOTSTRAPPING AND JACKKNIFING 159 12 4 1 Consensus trees A consensus tree provides an estimate for the level of support for each clade in the final tree It is built by combining clades which occurred in at least a certain percentage of the resampled trees This percentage is called the consensus support threshold A 100 support threshold results in a Strict consensus tree which is a tree where the included clades are those that are present in all the trees of the original set A 50 threshold results in a Majority rule consensus tree that includes only those clades that are present in the majority of the trees in the original set A threshold less that 50 gives rise to a Greedy consensus tree In constructing a Greedy consensus tree clades are first ordered according to the number of times they appear i e the amount of support they have then the consensus tree is constructed progressively to include all those clades whose support is above the threshold and that are compatible with the tree constructed so far The length of the consensus tree branches is computed from the average over all trees contain ing the clade The lengths of tip branches are computed by averaging over all trees Note The above definitions apply to rooted trees The same principles can be applied to un rooted trees by replacing clades
112. aximum Hits 100 3 Y More Options Cancel Search Figure 15 1 BLAST Options e Discontiguous Megablast A variation on blastn that is slower but more sensitive It will find more dissimilar matches so it is ideal for cross species comparison e blastp Compares an amino acid query sequence against a protein sequence database P pP query seq 8 P q e blastx Compares a nucleotide query sequence translated in all reading frames against a protein sequence database You could use this option to find potential translation prod ucts of an unknown nucleotide sequence tblastn Compares a protein query sequence against a nucleotide sequence database dy namically translated in all reading frames tblastx Compares the six frame translations of a nucleotide query sequence against the six frame translations of a nucleotide sequence database Three options are available for displaying your results e Hit table Returns one alignment for every hit against the database and displays them in a hit table Each query displays a separate table and is also viewable as a query centric alignment This is suitable for less than 100 queries 15 2 BLAST RESULTS 203 e Query centric alignment Returns one alignment for each query showing the hits aligned against the query sequence This is well suited for large batch searches but it doesn t dis play a hit table e Bin into has hit vs no hit Returns two sequence lists o
113. base Custom BLAST 15 1 Setting up a BLAST search To run a BLAST search in Geneious select your query sequence or sequences and click the BLAST button in the toolbar This operation can also be accessed by going to the Tools menu or by right clicking Ctrl click on Mac OS X on a sequence document and choosing BLAST Geneious gives you the option of searching against a database using either your currently se lected sequence documents or a sequence you enter manually If you choose to enter your sequence manually then Geneious will display a large text box in which you can enter your query sequence as either unformatted text or FASTA format Select your database using the first drop down box Databases are grouped together under their respective services Then choose which kind of BLAST search you wish to run under Program The available programs will depend on the database you have chosen Geneious can perform seven different kinds of BLAST search e blastn Compares a nucleotide query sequence against a nucleotide sequence database e Megablast A variation on blastn that is faster but only finds matches with high similarity 201 202 CHAPTER 15 BLAST 600 Sequence Search Orangutan Query Selected region _ Enter unformatted or FASTA sequence Database nr Y Add Remove Databases Program Megablast fast high similarity sal Results Hit table j a Retrieve Matching region M
114. ble in Geneious can be customized to suit your work Customize Feature Set Reset All Preferences Apply Cancel ok Figure 1 4 The plugins preferences in Geneious 18 CHAPTER 1 GETTING STARTED e Also check for beta releases of plugins Plugins are sometimes initially released as a beta for the purposes of testing before the officially release Check this to be notified about the release of beta plugins Customizing features To see a list of all the features in Geneious click Customize feature set Features can be turned on or off by checking or un checking the Enabled box next to each feature You might like to turn off the Tree Builder and Tree Viewer plugins if you don t do phylogenetics for example 1 3 Upgrading to new versions To upgrade existing Geneious installations simply download and install the new version to the same location Your existing data and license will be automatically loaded in the new version If you are upgrading to a new major version e g 8 0 to 8 1 or 8 1 to 9 0 Geneious will update your data folder to the new format creating a new folder with the upgrade s version number in the name and will offer to keep a copy of your old data folder in case you wish to downgrade 1 3 1 Downgrading to an earlier version If you chose to keep a copy of your old data folder when you upgraded you can easily down grade if you prefer to use the earlier version or if your license
115. ce for the alignment or assembly the annotations are added to the consensus sequence The results are also displayed in the annotations table and the following columns can be dis played e Change Indicates the reference sequence nucleotides followed by the variant nucleotides For example C A e Coverage The number of reads that cover the SNP region in the contig The coverage includes both the reads containing the SNP and other reads at that position Reference Frequency The percentage of reads that agree with the reference sequence at that position This field will only be present if at least 1 read agrees with the reference sequence e Variant Frequency The percentage of reads that have the variation at that position For variations that span more than a single nucleotide the variant frequency may appear as a range e g 47 8 51 7 to indicate the minimum maximum variant frequency over that range Polymorphism Type This may be one of the following SNP Transition a single nucleotide transition change from the reference sequence SNP Transversion a single nucleotide transversion change from the reference sequence SNP At a single position there are multiple variations from the reference sequence Substitution A change of 2 or more adjacent nucleotides from the reference sequence Insertion 1 or more nucleotides inserted relative to the reference sequence Deletion 1 or more nucleotides deleted relative to the
116. ces 5 2 4 Genome View The genome view Figure 5 3 is displayed when sequences larger than 100 000 bp are selected either as individual sequences or within a sequence list The genome viewer contains additional controls which allow for the efficient navigation of large sequences 1 The Go to Position button allows for the instant navigation to a particular nucleotide coordinate for any sequence in the current document selection It also allows the selection of a particular region of sequence or the selection of particular sequences out of a sequence list 2 A minimap is shown above the sequence viewer which shows a representation of the entire sequence plus its underlying annotations The portion of the sequence currently visible in the 66 CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES Annotations Virtual Gel Lengths Graph Text View History Notes Er Extract GRC A Translate VP Allow Editing Add Edit Annotation lg Annotate amp Predict gt 90 100 000 150 000 ATMGO0920 1 30 250 000 253 366 254 000 ChrM v Microsatellites Pentanucleotide Repeat hh b Trinucleotide Repeat an Dinucleotide Rey eat lt Q cooo v Known Genes Gia Dinucleotide Repeat GR Tetranucleotide Repeat exon y microRNA ATMG00930 1 4 ATMG00950 1 4 Prediced ORF y J E E ee 100 GC Content 50 0 Figure 5 3 The minimap and sequence view of a chromosome with gene and variation anno tations under the genome view
117. code and preferred codon usage of a particular expression host The resulting sequence is optimized to avoid use of codons that rarely occur in the highly expressed genes of the expression host thus increasing its expression level when cloned into that species In addition the resulting sequence can be forced to avoid cleavage sites for a set of restriction enzymes that you specify This tool calculates the Codon Adaptation Index CAI of a gene sequence as the geometric mean of the relative adaptiveness w of the codons in the sequence as defined in Sharp and Li 1987 Relative adaptiveness is calculated from Codon Usage Tables supplied by EMBOSS 14 9 OPTIMIZE CODONS 199 which calculate the relative codon usage of a selected set of genes in the target organism The optimized sequence returned is the one for which CAI is maximised You can configure the following options Figure 14 7 Annotate Codons creates an annotation track on the existing sequence with an annotation on each codon that is changed in the target sequence Each annotation has qualifiers that specify the original codon the replacement codon and the relative adaptiveness value W of the replacement codon calculated for the selected target organism codon utilization table The annotation track contains a qualifier with the CAI value of the result sequence Optimize and Save As creates a new sequence document that contains the result codons and the same annotation track
118. cost Enter the desired gap costs for the alignment Free end gaps Select this option to avoid penalizing gaps at either end of the alignment See details in the Pairwise Alignment section above Preserve original sequence order Select this option to have the order of the sequences in the table preserved so that the alignment contains the sequences in the same order Additional options Any additional parameters accepted by the ClustalW command line program can be entered here Refer to the ClustalW manual for a description of the avail able parameters Custom ClustalW executable The ClustalW2 executable is bundled with Geneious so there is no need to download this yourself However if you wish to use a different executable from the one within Geneious you can choose this here You can also do a Clustal alignment via translation and back see section 9 2 6 After entering the desired options click OK and ClustalW will be called to align the selected sequences or alignment Once complete a new alignment document will be generated with the result as detailed previously 9 2 4 Sequence alignment using MUSCLE MUSCLE is public domain multiple alignment software for protein and nucleotide sequences MUSCLE stands for multiple sequence comparison by log expectation 9 2 SEQUENCE ALIGNMENTS 117 To perform an alignment using MUSCLE select the sequences or alignment you wish to align and select Align Assemble Multiple Align Sele
119. ct MUSCLE as the alignment type and the options available for a MUSCLE alignment will be displayed For more information on muscle and its options please refer to the original documentation for the program http www drive5 com muscle muscle html 9 2 5 Other sequence alignment plugins for Geneious MAFFT MAFFT Multiple Alignment using Fast Fourier Transform is a fast multiple alignment pro gram suitable for large alignments To use MAFFT you must first download the plugin by going to Plugins under the Tools menu and selecting MAFFT Multiple Alignmen from the list of available plugins Click the Install button to install it and then click OK to close the Plugins window To run MAFFT select the sequences or alignment you wish to align select the Align Assemble button from the Toolbar and choose Multiple Alignment MAFFT should now be showing as an option for the type of alignment For more information on MAFFT and its options please refer to the original documentation for the program http mafft cbrc jp alignment software Mauve The Mauve aligner allows you to construct whole genome multiple alignments in the presence of large scale evolutionary events such as rearrangement and inversion To use Mauve you must first download the plugin by going to Plugins under the Tools menu and selecting Mauve from the list of available plugins Click the Install button to install it and then click OK to close the Plugins window To run Mauve
120. ction For junctions of type Fusion the junction site is split into two separate annotations each with a jagged edge on one side of the interval to indicate the side which jumps elsewhere e Intron Size This is present when Junction Type is Intron Reads supporting discovery Indicates the number of reads that supported discovery of this junction during the first pass This may be lower than the advanced minimum support setting in cases where other reads supported discovery of a slightly offset version of this junction which allows this junction to be retained on the next pass e Reads using Indicates the number of reads that used this junction as part of their map ping during the second pass Junction Source amp Junction Destination Clickable links to the junction positions in the reference sequence When the destination is a different reference sequence this is prefixed with the sequence name followed by a colon e Color Annotations are colored from blue to green based on increasing values of Reads supporting discovery Reads spanning junctions may be represented in one of two possible ways For introns under 50 bp the intron is represented as a gap in the read For longer introns or for fusion genes two copies of the read appear in the contig where the fragment of the read extending past the junction is marked as trimmed 10 3 5 The map to reference algorithm The reference assembly algorithm used is a seed and expand style ma
121. cument s Step You can create branches in your workflows by using the Save Documents Branch Step often in conjunction with a Filter Documents Step as the first Step in each branch For an example on branching and filtering see the sample Identify Organism Workflow 16 3 Custom code in Workflows Custom code allows you to create Geneious operations that do almost anything The Work flow custom code automatically inserts the surrounding import statements for the complete Geneious API and a class framework around the methods you implement here Additional import statements can be provided prior to the first method Documentation for the API is available at Geneious API For more advanced programmatic access to Geneious for example creating importers exporters or viewers please download and refer to the Geneious Plugin Development Kit Chapter 17 Geneious Education This feature allows a teacher to create interactive tutorials and exercises for their students A tutorial consists of a number of HTML pages and Geneious documents The student edits the pages and documents to answer the tutorial questions and then exports the tutorial to submit for marking 17 1 Creating a tutorial Geneious Tutorials are comprised of HTML documents with linked images and geneious files Simply create your html documents and place them together in a folder The first page of the tutorial should be called index html and this will be loaded as t
122. cut sites on a sequence Shift click on the two restriction site annotations in the sequence view 14 2 Digest into fragments The option Digest into fragments from the Tools Cloning menu or the context menu allows you to generate the nucleotide sequences that would result from a digestion experiment You can digest multiple nucleotide sequences at a time If the digestion results in overhangs these will be recorded as annotations on the fragments 184 CHAPTER 14 CLONING Y Minimum recognition sequence length 4 nucleotides C Only consider enzymes with palindromic recognition sequence GACGTAC P 3 4 nucleot CpG EcoBl per match GTAMKAC P 5 2 nucleot CpG per match AGACT 4 blunt EcoBl per match GATGCAC a 5 4 nucleot CpG EcoKI per match CAYCGRG 5 4 nucleot CpG per match GAGWCC 4 5 3 nucleot Dcm CpG per match TGGACCA A blunt Dcm per match GAGATCC A 5 4 nucleot Dam Dem per match GCCNNNNA 6 3 nucleot Dcm CpG per match AAGATCT 4 nucleot Dam EcoBl per match GAGTNACC 5 nucleot Dcm CpG E 1 per match CCAWGG 1 nucleot Dcm per match GCGAC 2 nucleot CpG per match ATACGAT 2 nucleot Dam CpG E 1 per match TTTAAAA blunt EcoKI per match GAAATTC 5 4 nucleot CpG EcoBl per match ACCWGG 5 5 nucleot Dem per match GATAATC blunt per match RGCGCAY 3 4 nucleot Dcm CpG E 1 per match GGACC blunt Dcm CpG per match GCGAC 3 2 nucleo
123. d connection settings install plugins customize the appearance and behaviour of Geneious define shortcut keys and set up sequencing profiles Many of these options are explained in more detail in the next sections The tabs in the Preferences window are as follows General This tab contains general setup options Data storage location shows the location of your Geneious database see section 1 2 2 1 2 GENEIOUS SETUP 11 Search history allows you to clear your search history e Check for updates Geneious can check for the release of new versions every time it is started If a new version has been released Geneious will tell you and give you a link to download it Geneious will also check for beta versions if you have enabled this option A beta version is a version that is released before the official release for the purposes of testing It may therefore be less stable than official releases e Max memory available to Geneious allows you to change the RAM allocated to Geneious see section 1 2 4 e Advanced allows editing of advanced preferences You should not alter these unless you know what you are doing e Connection settings set how Geneious connects to the internet see section 1 2 5 Plugins and Features Install plugins and customize features in this tab see section 1 2 6 This tab can also be accessed via Tools Plugins Appearance and Behavior In the Appearance panel you can change the way the m
124. d in the status bar directly below the toolbar To exit the search window and return to browsing click the orange X in the search options bar or press the Escape key while the cursor is in the search text field Important You must use quotation marks if and blank spaces are part of your search criteria No quotation marks lead to unreliable results Autocompletion of search words Geneious remembers previously searched keywords and offers an auto complete option This works in a similar way to Google or predictive text on your mobile phone If you click within the search field a drop down box will appear showing previously used options 54 CHAPTER 4 MANAGING YOUR LOCAL DOCUMENTS T Immunodeficiency M Include Subfolders Y Whole Words Only Search Y More Options x Score Folder Name Description 4 R 91 Alignments S HIV env 20 HIV envelope genes 89 Alignments HIV env alignment Alignment of 19 sequences 89 Alignments HIV env alignment realigned Alignment of 19 sequences i 89 Alignments HIV env alignment partially realigned Alignment of 19 sequences Realigned between bases 91 Alignments Nucleotide alignment 2 Alignment of 20 sequences l 100 Alignments HQ625578 HIV 1 isolate SA C86_A9 from South Africa envelope g 100 Alignments Y HQ625578 translation HIV 1 isolate SA C86_A9 from South Africa envelope g 38 Genomes Viruses HIV Human immunodeficiency virus 1 comp
125. de all inactively linked documents as well as those documents parents or descendants This means that you will only be viewing documents that are directly affected by one currently being viewed You can reactivate temporarily deactivated links from the view by right clicking Windows Linux or control clicking MacOS on a document and choosing Activate link to parent from the context menu Alternatively you can reactivate links to all children at once by choosing Show Operations and right or control clicking then selecting Reactivate all links for this operation You may also manually deactivate links in this fashion Figure 6 7 Sequence View Annotations Dotplot Self Virtual Gel DNA Fold Enzymes Fragments Text View Ma bg Ol E properties M Show Operations M Show Inactive Links 2 Go to Export J History Lineage Parents Descendants O TetR repressiple GFP generator inserted into pSB1C3 O TetR repressiple GFP generator inserted into pSB1C3 v EB Restriction Cloning Today at 3 43 PM v BB Extract PCR Product Today at 3 45 PM O psB1C3 E Terr repressiple GFP generator inserted into pSB1C3 PCR Produc a 2 Go to Activate link to child reruns operation TT Activates the link between this document and its child Changes to the parent will be propagated to the child TT Figure 6 7 Context Menu Note reactivating links immediately reruns the operation depending on the size and type of the operati
126. des you can further restrict your search with the second drop down box e is greater than gt e is less than lt 4 2 SEARCHING AND FILTERING LOCAL DOCUMENTS 55 e is greater than or equal to gt e is less than or equal to lt Likewise if you are searching on the Creation Date search field you have the following op tions e is before or on e is after or on e is between When searching your local folders you have the option of searching by Document type The second drop down list provides the options is and is not The third drop down lists the various types of documents that can be stored in Geneious such as 3D Structure Nucleotide sequence and PDF see Figure 4 2 Match _ all a of the following Document type BJ is y 3D structure H oP M include Subfolders Fewer Options 3 Name Summary R af Alpha Helix Alpha Helix ag Cyclopropane Cyclopropane af VirusVIRAL PROTEIN VirusVIRAL PROTEIN 1G5G aaa a gt Figure 4 2 Document type search options And Or searches The advanced options lets you search using multiple criteria By clicking the button on right of the search term you can add another search criteria You can remove search criteria by clicking on the appropriate button The Match all any of the following option at the top of the search terms determines how
127. e In Geneious R8 and higher on Windows and Linux machines an appropriate amount of RAM based on your total RAM will automatically be allocated In Geneious R7 and earlier and on all mac versions the default memory allocation is 1GB of RAM on 64 bit machines and 700MB of RAM on 32 bit machines To change the amount of memory allocated to Geneious go to Tools Preferences and in the General tab increase the Max memory available to Geneious setting On Windows you need to run Geneious as an administrator to change this setting close Geneious then right click on the Geneious icon and choose Run as administrator On a 32 bit machine you can only allocate a maximum of 1GB so if you need more RAM than this you ll need a 64 bit machine You should never allocate the total memory of your computer to Geneious as you need to leave some RAM available for your operating system As a general rule of thumb on a 64 bit machine with up to 8GB of RAM in total you can allocate half the RAM to Geneious On machines with more than 8GB RAM you should leave 3 4GB spare for your operating system and allocate the rest to Geneious To see how much memory Geneious is using check the Memory Usage Bar under the Sources panel You may need to turn this on under Tools Preferences Appearance and Behavior Clicking this bar will force a garbage collection and free up memory within the JVM 1 2 5 Connecting to the internet from within Geneious In order
128. e Graphs tab and the other options will show up under the Annotate and Predict menu Further information on these applications is available at http emboss open bio org 8 3 Compare Annotations Geneious can compare annotations across up to 3 annotation tracks or documents highlighting annotations which are common or unique depending on which criteria you choose To use this function select the sequence or sequences containing annotations you wish to com pare and go to Annotate and Predict Compare Annotations In the Annotation Types panel select the annotations you wish to compare The default setting is for pairwise comparison if you wish to do three way comparison select Set C Choose the annotation type and location for each set and the type of comparison Comparison options are e Names must match The names of the annotations must be the same When allowing partial matches for polymorphism annotations just the name of the matching region of the annotation must match 8 3 COMPARE ANNOTATIONS 105 All properties must match All properties in addition to the name must be the same to be considered a match e Allow intervals to partially match When one annotation partially overlaps another you can choose to return either Partial Annotations or Full Annotations Partial Annotations will return annotations spanning or excluding only the matching region Full Annota tions will return annotations spanning the entire length o
129. e Name column in Geneious e Export Folder To export an entire folder to a single file click on the folder in the Sources panel and go to File Export Export Folder Note that folders can only be exported in geneious format 3 6 Printing and Saving Images Geneious allows you to print or save as an image the current display for any document viewer This includes the sequence viewer tree view dotplot and text view 3 6 1 Printing Choose Print from the file menu The view is printed without the options panel It is recom mended to turn on Wrap sequence and deselect Colors before printing Wrapping prints the sequence as seen in the sequence viewer and the font size is chosen to fill the horizontal width of the page The following options are available Portrait or landscape Controls the orientation of the page Scale Can be used to decrease or increase the size of everything in the view while still printing within the same region of the page For many types of document views this will cause it to wrap to the following line earlier usually requiring more pages Size Controls the size the printed region on the paper Effectively increasing the size reduces the margins on the page 3 6 2 Saving Images Choose Save as image file from the file menu The following options are available 50 CHAPTER 3 IMPORTING AND EXPORTING DATA Size Controls the size of the image to be saved Depending on the document view being sa
130. e Sequence Project SwissProt Curated protein sequences information from EMBL You can also edit the databases that show up in Geneious by clicking on Edit Databases in the Set up BLAST Services window This will only change the databases that Geneious displays and will not have any effect on the actual databases on the BLAST server 15 4 Custom BLAST Custom BLAST allows you to create your own custom database from either FASTA files or sequences in your local folders and BLAST against it The Custom BLAST plugin requires access to NCBI BLAST binary files 15 4 CUSTOM BLAST 207 15 4 1 Setting up the Custom BLAST files through Geneious Geneious provides a download manager to help you download and extract the Custom BLAST files To use it go to Tools Add Remove Databases Set Up BLAST Services and select Custom BLAST from the Service drop down box see Figure 15 4 Make sure Let Geneious do the setup is checked Then click OK After a few seconds the compressed file containing all the files needed to run Custom BLAST will start downloading You can click Pause to pause the download You can add and search Custom BLAST databases as soon as it has finished downloading and extracting If you shut down Geneious with the file partially downloaded you will need to start downloading it again from the beginning 15 4 2 Setting up the Custom BLAST files yourself If you want you can download or otherwise acquire the NCBI BLAST
131. e Start end and length of the extraction or only the length if the whole sequence is used An Overhang is shown on one or both sides of the tag Only one overhang is shown if no over hang can or has to be created for the side with the missing overhang The position top bottom of the overhangs is an indication for whether it is a 3 or 5 overhang the color indicates a valid match an error state Errors between sequences e g multiples of the same overhang se quence will be shown below the insert field When Save intermediate products is checked the PCR products and digested sequences will be saved in addition to the final sequence where applicable 14 6 1 Sequence ordering rules and assumptions Geneious will analyze your backbone if defined and each sequence passed to it and will detect existing type IIS restriction sites overhang annotations primer annotations and blunt ends When Auto arrange is enabled Geneious will try to identify the correct sorting of the se quences based on the available overhangs The Auto arrange button will be lighter grey when enabled and darker grey when switched off If tags are reordered or reverse complemented manually Auto arrange will be switched off and Geneious will not try to determine the correct order Note that Auto arrange will not arrange based on sequence names 194 CHAPTER 14 CLONING Geneious will use the existing restriction sites overhang annotations and primer annotation
132. e a personal license or choose to connect to a license server The options are as follows e Use license key If you have purchased a personal license you can enter the details here to activate it Make sure you enter the licensee name exactly as it appears in the email in which you received your activation ID registration key An internet connection is re quired to activate personal licenses and you may need to configure your firewall proxy settings to enable access to http licensing biomatters com on port 80 e Use license server If your organization has purchased a floating license administered through a FLEXnet license server this is where you enter the details required to connect to the license server Ask your system administrator for the host name and port of the license server e Use Sassafras KeyServer If your organization has purchased a floating license admin istered through Sassafras KeyServer select this option Your system administrator needs to configure KeyAccess to point to the KeyServer license server 1 4 2 Install FLEXnet This installs the FLEXnet license manager which is necessary for activating a personal license This is normally installed automatically when Geneious is installed but Geneious will tell you if need to run this when you activate your license Only an administrator on your computer can do this but it only needs to be done once from one user account Once this has been done any non admin user can ac
133. e failure and attempt to be as specific as possible about the cause of the failure Figure 6 1 Inactive links do not propagate changes from parent to child Inactive links are created in two different ways firstly when you choose not to propagate changes that active link becomes temporarily inactive Secondly if an operation does not support creation of active links or was told not to create them all links between its parents and children will be permanently inactive 85 86 CHAPTER 6 PARENT DESCENDANT TRACKING fe A Descendant changes were not successfully applied for 1 operation s although the O parent document was successfully saved The failed operation s have been changed to inactive links Cause Cannot identify forward primer to use in extraction For help with this issue dick Show Details then contact support with the details included Y Show details Figure 6 1 Failure to propagate an Extract PCR product operation due to a missing forward primer All operations in Geneious at least create inactive links The following operations in Geneious can produce actively linked documents e Cloning Digest into Fragments e Cloning Insert into Vector e Cloning Ligate sequences e Cloning Gateway e Primers Extract PCR product e Sequence Viewer Extract e Sequence Viewer Translate Note Extract and Translate will not create active links by default To do so you must select the Active
134. e labels This will reduce screen clutter by removing annotation labels which are too frequent Sizes Here you can set the font size for bases labels names and numbering 5 2 9 Statistics The Statistics tab displays statistics about the sequence s being viewed If only part of the sequence alignment or assembly is selected then the statistics displayed will correspond to the highlighted part The length of the sequence or part of the sequence is displayed next to the Statistics option e Residue frequencies This section lists the residues and their frequencies for both DNA and amino acid sequences for both single sequences and alignments assemblies It gives the frequency of each nucleotide or amino acid over the entire length of the sequence includ ing gaps If there are gaps then a second percentage frequency is calculated ignoring gap characters The G C content for nucleotide sequences is shown as well for easy reference see GC content below 5 2 THE SEQUENCE VIEWER 73 e Amino acid and codon frequencies These are listed for nucleotide sequences based on the current translation options Click Options to change the translation options For codon usage statistics the frequency of all 64 codons with their associated amino acid will be displayed If any CDS contains non standard start codons then some of the 64 codons may be split into 2 entries based on whether they translate to methionine or their standard translation
135. e sequence and you are interested in finding differences between each sequence and the reference or SNPs 11 1 1 Find Variations SNPs Manually investigating every little disagreement can be time consuming on larger contigs The Find Variations SNPs feature from the Annotate Predict menu will annotate regions of dis agreement and can be configured to only find disagreements above a minimum threshold to screen out disagreements due to read errors This feature can also be configured to only find disagreements in coding regions if the reference sequence has CDS annotations present and can analyze the effects of variations on the protein translation to allow you to quickly identify silent or non silent mutations It can also calculate p values for variations and filter only for variations with a specified maximum P Value For full details of how the various settings work in the Variation SNP finder hover the mouse over them in Geneious to read the tooltips or click one of the buttons 145 146 CHAPTER 11 ANALYSIS OF ASSEMBLIES AND ALIGNMENTS P values The p value represents the probability of a sequencing error resulting in observing bases with at least the given sum of qualities The lower the p value the more likely the variation at the given position represents an real variant Click the down arrow next to the exponent of the Maximum Variant P Value setting to increase the number of variants found When calculating P Values
136. e sequence name and press the delete key To delete a region of a sequence or alignment select the region and press the delete or backspace key Normally this will move residues on the right into the deleted area To move the residues on the left into the deleted area hold down the Shift key while deleting To drag a sequence to the left or right select the region you want to move then click it again and drag it to the position you want Dragging will either move residues over existing gaps or open new gaps when necessary Dragging a selection consisting entirely of gaps moves the gaps to the new location After editing is complete click Save to permanently save the new contents 5 3 1 Concatenating sequences To join several sequences end on end select all the sequences and go to Tools Concatenate Sequences or Alignments This creates a single sequence document from the input sequences The order in which sequences are concatenated can be chosen in the setup dialog box and the resulting sequence can be circularized if required by checking Circularize sequences If one or more of the component sequences was an extraction from over the origin of a circular sequence you can choose to use the numbering from that sequence thus producing a circular sequence with its origin in the same place as the original circular sequence Overhangs will be taken into account when concatenating 5 4 COMPLEMENT AND REVERSE COMPLEMENT 77 5 4 Complemen
137. e server can see anything in there without having to get it from your Geneious client This means large documents such as NGS sequencing data can be placed in here and the server will be able to quickly access it Also if you log into the server from another machine documents you put in the Shared Database will be available unlike those of your local database You can also see other users data so this is a good way to share your documents This is exactly like the normal shared Databases available with Geneious but this database is preconfigured and available as soon as you log into the server Don t try and access it any other way using the normal shared Database plugin 21 3 RUNNING JOBS AND RETRIEVING RESULTS 235 You are not currently logged in to Geneious Server Login en Log in to Geneious Server J Use SSL Port 8080 5 E Gene B Genome Nucleotide B PopSet E Protein E PubMed snp Structure e Taxonomy User Name Password Save password Cancel ok _ gt z _n _ Figure 21 2 Log in to Geneious Server 21 3 Running jobs and retrieving results Once you ve logged into Geneious Server many normal operations will now include an addi tional pair of buttons indicating whether the job should run on your computer or on Geneious Server Figure 21 3 Whenever you see this choice you can choose to run the job on Geneious Server If you re not logged in when
138. e these tracks select the reference sequence document and go to Annotate amp Predict Compare Expression Levels Like Calculate Expression Levels which must be run on all contigs from a single sample at once to produce correct results in all cases Compare Expression Levels should also be run on all reference sequences at once However Compare Expression Levels can optionally be run on just one reference sequence at a time when using a normalization system other than Median of Gene Expression Ratios Values to Compare Either read counts fragment counts or transcript counts from each annotation can be com pared Since a single transcript can produce multiple reads and fragments the number of reads and fragments produced aren t independent events so the confidence values produced by comparing these are unlikely to be accurate For this reason we recommend comparing samples using transcript counts Normalization Since different samples produce different quantities of transcripts in order to compare values between samples the counts need to be normalized using one of the following methods Total Count The counts in each gene are scaled according to the total number of tran scripts mapped to all genes For example if one sample has twice as many transcripts mapped as the other sample then the counts for each gene need to be halved to make them comparable with the other sample e Median Expression The expression level of all e
139. e under the Cloning menu e Find Restriction Sites finds and annotates restriction sites on your sequence of interest from a candidate set of restriction enzymes See section 14 1 Digest into fragments allows you to generate the actual fragments that would be cre ated in a digestion experiment using restriction enzymes See section 14 2 e Ligate Sequences lets you ligate two or more fragments with or without overhangs e Find non cutting enzymes returns a list of enzymes which do not cut your sequence e Restriction Cloning allows you to choose a digested fragment or a sequence with two restriction site annotations to use as an insert and insert them into a vector circular sequence Geneious can do the work of working out what cut sites on the vector are compatible with the overhangs on the insert with some additional information from you See section 14 3 Gibson Assembly provides a one step interface to perform a Gibson Assembly or sim ilar operation a isothermal ligase independent restriction free overlap extension PCR cloning You can chose to insert one or multiple inserts into one or multiple vectors and specify the insertion order The operation automatically creates the necessary primers and the products you will get and generates a report document See section 14 4 e Gateway Cloning automatically detects Att sites on the insert and vector s and per forms a one step Gateway Cloning reaction See s
140. eate a new sequence document containing the selected sequence 5 1 1 Sequence lists Sequence lists make it easier to manage large numbers of sequences by grouping related se quences into a single document When you import files containing multiple sequences you will be asked if you want to store those sequences in a list To existing sequences in your database into a list or combine two lists into one select the sequences or sequence lists you want to group and go to Sequence Group Sequences into a List Note that this copies your sequences into a list and retains the original sequence documents To extract sequences from a list select the sequence s you want to extract and go to Sequence Extract Sequences from List This will copy each sequence out of the list into a separate 61 62 CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES 1 73 TTATCCACCAGTGGTTTTAGAGAGAATAATGTGGAGTTCCTATATAGGTTTGACATTATAAATCACTTCTTGA Name Sequence 1 Description Organism Type Nucleotide 4 Characteristics C Circular Cancel ok Figure 5 1 Entering a new sequence in Geneious document while retaining the original sequence within the list To remove a sequence from a list entirely select it and click the Delete button on your keyboard or go to Delete under the Edit menu 5 2 The Sequence Viewer Sequences are displayed in the viewer below the document table Annotations Chapter 8
141. ected sequences meet the overlap requirements Note The orientation of fragments will be determined automatically and they will be reverse complemented where necessary If you already have a contig and you want to add a sequence to it or join it to another contig then just select the contig and the contig sequence and click de novo assembly as normal Scaffolding Scaffolds are contigs which are linked together with the missing regions between them filled by Ns The size of the missing region is based on paired read distances The Geneious assembler will produce scaffolds if this option is turned on under More options If this setting is disabled it is because your data does not have paired reads or you haven t marked the data as paired using Set Paired Reads from the Sequence menu Unlike some assemblers where scaffolding is performed after contig formation Geneious scaf folding is integrated into the contig assembly process When there is strong support for scaf folding it may take precedence over potentially conflicting standard contig formation For this reason Geneious can t be configured to produce both scaffolds and non scaffolds from a single run 10 2 DENOVO ASSEMBLY 135 De novo assembly of circular genomes The Geneious de novo assembler can produce a circular contig if the ends of a contig match To enable this option click the More Options button and check Circularize contigs with matching ends Circularization requ
142. ection 14 5 181 182 CHAPTER 14 CLONING e Golden Gate simulates the digestion and ligation of multiple sequences with the same Type IIS enzyme in a single reaction See section 14 6 e TOPO cloning automatically detects TOPO vectors amongst the selected sequences and inserts the fragments into these vectors using a BLUNT TA or Directional Cloning approach See section 14 7 e Find CRISPR sites annotates specific CRISPR guide sequences in your sequence of interest and scores them based on offsite targets in your specified genome See section 14 8 The following sections give more detail on each option 14 1 Find Restriction Sites Restriction Enzymes cut a nucleotide sequence at specific positions relative to the occurrences of the enzyme s recognition sequence in the sequence For example the enzyme EcoRI has the recognition sequence GAATTC and cuts both the strand and the antistrand sequence after the G inside the recognition sequence leaving a single stranded overhang sticky end overhang GAATTC CTTAAG The option Find Restriction Sites from the Tools Cloning menu or the context menu allows you to find and annotate restriction sites on a nucleotide sequence You can configure the following options e Candidate Enzymes lets you select a set of restriction enzymes from which you want to draw the ones to use in the analysis This includes the options to use commonly used or all known commercially availab
143. ee building in Geneious 12 3 Tree building methods and models 12 4 Resampling Bootstrapping and jackknifing oaoa 12 5 Viewing and formatting trees PCR Primers 13 1 Design New Primers 13 2 Manual primer design 100 105 105 107 114 118 121 121 128 132 136 139 139 141 141 144 149 149 150 152 154 156 6 CONTENTS 13 3 Importing primers from a spreadsheet 4544454 eo wee ee we es 168 134 Primer Databases a sk es EK PRE EK Eee SNARES HEL ERS LE KS 170 135 Testwith Sayed Primers oo e ea ro AAA Ci it ewes 171 13 6 Characteristics lor Selecion oo ciosa dr ARA da RA RG 172 Es 3 1 oee e ee Ae he a a Se ee ee TEDA eS 172 LES Primer Extensions o a sae bw oe A A A A OP a es 173 BO Extat FCR POGUE y i a a eh ke S EA AE id 173 13 10More i BO oe aed td A a a EES 175 14 Cloning 177 TLI Find Tees ici Sies o soa soei ER 178 14 2 Digest Dto PAPERS ee eed ee ee aaa eee ed EY 179 A Teese res sk ea RAE A A WE Ee aS 182 16s Gbon ACO ae dk eA eee AE A OE a RY 185 145 Gateway Cloning ee ee ee ds ee ee OES OE ORES ES 187 146 Golden Gale e eb bo we eee a a eS Se ela ha E lw ee eee wah 188 u7 TOPO Ce soon te ee ee ee Oe eek oes ee Be eS 191 14 8 CRISPR sile finder os i ee eK AOE PEE OE Pew dL ORE Ce RE 191 H9 OPI OS ide A AAN ee AA ee ee ee S 194 15 BLAST 197 15 1 Setting tip a BLAST search lt lt 0 00 be AA AA AAA 197 154 BLAST TUS o nees ee oS ee he AE A RA RA ES 199 150 ME GN
144. ees in Geneious see Section 12 5 12 2 Tree building in Geneious To build a tree select an alignment or a set of related sequences all DNA or all protein in the Document table and click the Tree icon or choose this option from the Tools menu If you are building a simple tree Neigbour joining or UPGMA using the Geneious tree builder the tree can be built directly from a set of unaligned sequences as the alignment will be built as part of the tree building process For more advanced trees or if you wish to bootstrap your trees you must build an alignment first and use that as input for your tree You can also select an existing tree document which contains an alignment and build another tree from that as the alignment will simply be extracted from the existing tree and used build the new tree The following options are available in the tree building dialog for the Geneious tree builder For more information on these options see sections 12 3 to 12 4 e Genetic distance model This lets the user choose the kind of substitution model used to estimate branch lengths If you are building a tree from DNA sequences you have the choices Jukes Cantor HKY and Tamura Nei If you are building a tree from amino acid sequences you only have the option of Jukes Cantor distance correction Tree building method There are two methods under this option Neighbor joining and UPGMA e Outgroup Choose which sample to use
145. enes Genome Whole genome sequences Nucleotide DNA sequences PopSet sets of DNA sequences from population studies Protein Protein sequences PubMed Biomedical literature citations and abstracts SNP Single Nucleotide Polymorphisms Structure 3D structural data Taxonomy Names and taxonomy of organisms Entrez Gene Entrez Gene is NCBI s database for gene specific information It does not include all known or predicted genes instead Entrez Gene focuses on the genomes that have been completely sequenced that have an active research community to contribute gene specific in formation or that are scheduled for intense sequence analysis The Entrez Genome database The Entrez genome database has been retired For backwards compatibility Geneious simulates searching of the old genome database by searching the Entrez Nucleotide database and filtering the results to include only genome results The Entrez Nucleotide database This database in GenBank contains 3 separate components that are also searchable databases EST GSS and CoreNucleotide The core nucleotide database brings together information from three other databases GenBank EMBI and DDBJ These are part of the International collaboration of Sequence Databases This database also contains Ref Seq records which are NCBI curated non redundant sets of sequences The Entrez Popset database This database contains sets of aligned sequences that are the result of populat
146. ength 861 Interval 4 153 gt 3 293 Bases ATGAGTATTCAACATTICCGT gene bla note ampR confers resistance to ampicillin codon_start 1 product beta lactamase translation MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGA VLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPEL NEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIV VIYTTGSQATMDERNROQIAEIGASLIKHW NCBI Feature Key CDS Figure 8 1 Annotation properties and qualifiers displayed by mousing over an annotation 95 96 CHAPTER 8 WORKING WITH ANNOTATIONS 8 1 1 Viewing and Customizing Annotations The Annotations and Tracks tab If a sequence contains annotations the Annotations and Tracks tab to the right of the sequence viewer will show a yellow arrow E and all the annotation types present on the sequence will be listed in this panel see figure 8 2 Annotations that are directly on the sequence are listed first followed by annotations on tracks Tracks with only one annotation type will show a single listing whilst tracks with multiple annotations will show a list of the annotation types Sequence View Annotations Virtual Gel Lengths Graph Text View History Notes BD S Extract E R C Translate 4 Allow Editing p Add Edit Annotation fi Annotate amp Predict 2 Primer Design Save eg 1 n 50 000 150 000 200 000 2
147. enses can only be released twice in a 6 month period so do not do it unnecessarily If you re using a floating license you can release it allowing another user to access it without you having to shut Geneious down Once you ve released the license Geneious will enter restricted use mode 1 4 5 Buy Online This item will open the Geneious store in your browser where you can upgrade licenses pur chase new licenses or contact our sales team 1 5 Troubleshooting For help with troubleshooting or to request a feature please contact the Geneious support team You can do this either by clicking the Support button located in the toolbar in Geneious or going to our support website at http support geneious com and submitting a request Clicking the Support button will automatically send through some system information to help us assist you If you are submitting a request through our website please include details of your operating system and the version of Geneious you are using and as much information as possible about the nature of your problem including screenshots to illustrate the issue if appropriate The Geneious support website also contains a comprehensive Knowledge Base with solutions to common problems and tips for getting the most out of Geneious as well as a User Forum where you can post questions to the Geneious community You can access the support website and download user manuals license agreements and re lease notes
148. equences This type of alignment is appropriate when aligning two segments of genomic DNA that may have local regions of similarity embedded in a background of a non homologous sequence A global alignment is a sequence alignment over the entire length of two or more nucleic acid or protein sequences In a global alignment the sequences are assumed to be homologous along their entire length Scoring systems in pairwise alignments In order to align a pair of sequences a scoring system is required to score matches and mis matches The scoring system can be as simple as 1 for a match and 1 for a mismatch between the pair of sequences at any given site of comparison However substitutions inser tions and deletions occur at different rates over evolutionary time This variation in rates is the result of a large number of factors including the mutation process genetic drift and natural selection For protein sequences the relative rates of different substitutions can be empirically determined by comparing a large number of related sequences These empirical measurements can then form the basis of a scoring system for aligning subsequent sequences Many scoring systems have been developed in this way These matrices incorporate the evolutionary prefer ences for certain substitutions over other kinds of substitutions in the form of log odd scores Popular matrices used for protein alignments are BLOSUM and PAM matrices Note The BLOS
149. er e Save Current State allows you to save the the current state if the columns so you can easily apply it to other tables You can give the state a name and it will then appear in the Load Column State menu e Load Column State contains all of the columns states you have saved Selecting a column state from here will immediately apply that state to the current table and lock the columns to maintain the new state Use Delete Column State to remove unwanted columns states from this menu Note New columns can be added to the document table by adding Meta Data to documents see section 5 7 Meta Data 2 3 The Document Viewer Panel The Document Viewer Panel shows the contents of any document clicked on in the Document Table allowing you to view sequences alignments trees 3D structures journal article abstracts and other types of documents in a graphical or plain text view Figure 2 3 Options for con trolling the look and layout of a given document are displayed in the right hand panel These options vary depending on what type of document you are viewing For detailed information on specific types of viewers please refer to the sections below Sequence Alignment Viewer section 5 2 3D Structure Viewer section 7 2 Dotplot Viewer section 9 1 Tree Viewer section 12 5 Journal Article Viewer section 3 3 3 To view large documents you can open them in a new window by double clicking In the document viewer panel th
150. er Steps in the workflow e Access exposed options via button Rather than displaying all the options for this work flow Step in the top level options dialog instead provide a button for the user to click on before showing the options for this Workflow Step Exposing options with dependencies Some options have dependencies on other op tions For example when a checkbox is off another option may become disabled If you choose to expose a subset of the options rather than all options the dependencies be tween options will be discarded For checkbox options that have an associated value you will probably want to expose the associated value too Often this associated value doesn t have a label in the user interface so it will appear with its programmatic name immediately following the checkbox option in the drop down list of available options Note that for some Operations not all options may be available when run in a Workflow Some special Steps also have configurable options which in some cases can also be exposed when the Workflow is running For example the Filter Documents Step can be exposed so that the user can set the Filter criteria when the Workflow is running 16 2 2 Advanced document management Grouping and separating documents In simple Workflows all documents provided to the Workflow are grouped as a single set of documents which are used as input to a single invocation of the first Step in the Workflow Each Workflow Step
151. er configuration 5 2 THE SEQUENCE VIEWER 67 viewing window is highlighted on the minimap showing the relative position of the visible section to the overall sequence The minimap can also be used to quickly navigate around the visible sequence Clicking on a section of the minimap will jump the sequence viewer to center on that position Double clicking the minimap will zoom further in on the clicked section Finally highlighting a section of the minimap using a click drag release action will display the highlighted region in the se quence viewer 5 2 5 The Side Panel Controls The panel to the right of the sequence viewer allows you to control what is displayed in the sequence viewer e g translations consensus sequences graphs and annotations displays sequence statistics and provides functions for finding annotations ORFs and restriction sites on your sequences A brief description of each tab is given below General Options Contains the color options see section 5 2 6 check boxes to turn on and off main aspects of the sequence view and options for what to display as the name of each sequence gt Display Contains options for displaying the translation and or complement of a sequence and turning off the original nucleotide sequence See sections 5 4 and 5 5 for more informa tion This tab is not displayed for protein sequences all Graphs This option is visible when viewing nucleotide or protein sequences chr
152. ere are two tabs that are common to most types of documents Text view and Info Text view shows the document s information in text format The exception to this rule occurs with PDF documents where the user needs to either click the View Docu ment button or double click to view it Under the Info tab you can view document Properties meta data section 5 7 History section 4 6 and Lineage section 6 2 2 4 THE HELP PANEL 25 BBD a Nucleotide sequence b Journal Article c Phylogenetic tree Figure 2 3 Three document viewers 2 3 1 General viewer controls There are several general options which are available on all viewers which are shown in the toolbar at the top right of the viewer Some of these can also be accessed through the View menu 24 Share Allows you to share the current visualization on Twitter Facebook or email 0 Split View Provides several options for splitting the view so that multiple views are shown simultaneously for one document When the view is split selection of annotations and regions of the sequence are synchronized across the viewers To close split views click the 4 button which is also on the right of the toolbar Expand View Expands the document view panel to fill the main window by hiding the sources panel on the right and the document table above Clicking this again will return the layout to it s original state O New Window Opens another view of the cur
153. erent rate to the transversions see Hasegawa et al 1985 Tamura Nei This model also assumes different equilibrium base frequencies In addition to distinguishing between transitions and transversions it also allows the two types of transitions A G and C gt T to have different rates see Tamura amp Nei 1993 12 3 4 Advanced Tree Building methods The following plugins are available for running maximum likelihood or Bayesian phylogenetic analyses in Geneious 158 CHAPTER 12 BUILDING PHYLOGENETIC TREES e MrBayes For Bayesian estimation of phylogenies runs MrBayes 2 0 9 http mrbayes sourceforge net e PhyML Builds maximum likelihood trees using PhyML 3 0 http www atgc montpellier fr phyml e GARLI Genetic Algorithm for Rapid Likelihood Inference Builds maximum like lihood trees from alignments of 4 or more sequences using Garli 2 0 https www nescent org wg_garli Main_Page RAxML Randomized Axelerated Maximum Likelihood Rapid maximum likelihood tree building using RAxML 7 2 8 http www exelixis lab org Allows parti tioned datasets e FastTree Approximately maximum likelihood phylogenetic trees from alignments of nucleotide or protein sequences using Fasttree 2 1 5 http www microbesonline org fasttree Ideal for large alignments e PAUP Builds maximum parsimony and maximum likelihood trees You must own or purchase a copy of PAUP to use this plugin http paup csit fsu edu
154. escribed in the keyboard shortcuts below e Select from annotations When annotations are available click on any annotation to se lect the annotated residues As with mouse dragging multiple selections are supported e Click on sequence name This will select the whole sequence e Select all Use the keyboard shortcut Ctrl A A on Mac to select everything in the panel Keyboard shortcuts for selection of sequences e To quickly select a single residue double click on it e To select a block of residues within a single sequence triple click e To select a block of residues across multiple sequences quadruple click e To select a block of 10 residues hold down Shift and Cntrl alt option on a Mac and press the keyboard arrow e To select a specific region of sequence click at beginning of the region you want to select hold down Shift and then click the end e To modify the right hand end of a selection hold down Shift alt command on a Mac and use the right left arrows to select more or fewer bases Holding down Shift and Cntrl alt option on a Mac modifies the selection by 10 bases at a time 5 2 THE SEQUENCE VIEWER 65 e To select the same region across multiple sequences in an alignment select the region you want in the first sequence then hold down Shift alt command on a mac and click the down arrow to apply the selection to the sequences underneath Holding down Shift Cntrl alt option on a mac while pressi
155. essages will only be sent and become visible to your contacts once you click Send or press the Enter key To leave the chat simply close the Chat Window 228 CHAPTER 19 COLLABORATION 19 5 3 Setting up and running your own Jabber server Setting up your own Jabber server is simple and means that your documents will never leave your local network This means that you will not have any problems with firewalls achieve much greater download speeds and it provides an extra security layer for the confidentiality of your documents Note the communication with the Geneious Jabber server is encrypted and that we do not log or share your data If you wish to set up and run your own Jabber server we recommend using Openfire from Ig nite Realtime http www igniterealtime org projects openfire index jsp which is available for free under the Apache 2 0 Open Source License http www apache org licenses LICENSE 2 0 html Install and start the server on one computer and then en ter that computer s name or address in the Server field under More Options when creating a new account Please note that Biomatters cannot provide any further support for setting up and managing your own Jabber server Chapter 20 Shared Databases A Shared Database allows you to store your documents in your favorite relational SQL database rather than on the file system This means that multiple users can concurrently use the same sync
156. ession Levels Counting The three metrics are calculated by normalizing the count of reads that map to each CDS anno tation If a read at least partially intersects at least one interval from a CDS annotation then it will be treated as though that read mapped to that CDS annotation For reads that map to multiple locations or reads that map to a location that intersect multiple CDS annotations these may either be counted as partial matches excluded from the calcula tions or counted as full matches to each location they map to For example if a read maps to two locations then it will be counted as if 0 5 reads mapped to each of the two locations When calculating statistics reads that don t map or map outside of an annotation CDS annota tion are ignored 11 2 ANALYZING EXPRESSION LEVELS 149 RPKM Reads per kilobase per million normalizes the raw count by transcript length and sequencing depth RPKM CDS read count 10 CDS length total mapped read count FPKM Same as RPKM except if the data is paired then only one of the mates is counted ie fragments are counted rather than reads TPM Transcripts per million as proposed by Wagner et al 2012 is a modification of RPKM designed to be consistent across samples It is normalized by total transcript count instead of read count in addition to average read length TPM CDS read count mean read length 10 CDS length total transcript count Resul
157. ets you hide atoms or change their size in the selected region of the molecule You can also choose whether to show hydrogen atoms and atom symbols Bonds lets you hide bonds or change their size in the selected region of the molecule Covalent ionic bonds hydrogen bonds and disulfide bonds can be affected separately Effects lets you toggle spin antialiasing stereo and slabbing effects for the whole molecule Save saves the current appearance of the molecule Chapter 8 Working with Annotations 8 1 Viewing editing and extracting annotations Annotations are used to describe and visualize features such as coding regions restriction sites and repetitive elements on sequences and alignments Annotations can either be annotated directly on a sequence in the sequence viewer or they can be grouped logically into tracks A track is a collection of one or more annotation types Tracks are stacked vertically underneath the sequence in question with a separate line for each track and its annotations An annotation may have one or more properties or qualifiers associated with them These can be added at the time an annotation is created or at a later date by editing the annotation To view the properties of a given annotation mouse over it in the sequence viewer This will display a tooltip listing the Name Type Length Interval and Sequence for that annotation plus any additional qualifiers see Figure 8 1 Name bla CDS Type CDS L
158. ew plugins are released Changes the way the program notifies you about new plugin releases 1 2 GENEIOUS SETUP 17 General AEE ETES Appearance and Behavior Keyboard NCBI Sequencing Plugins Plugins are downloadable modules which add new functionality to Geneious Available Plugins 4 Description Categories Y MrBayes Runs the well known program MrBayes included for Phylogenetics Mutate and Shuffle Automatically mutate or shuffle sequences Protein Nucl EMBOSS Protein Analysis Predict secondary structure antigenic Protein CpG Islands A nucleotide sequence graph for predicting CpG Nucleotide Contig Sorter Sorts sequences in a contig according to the Sequencing Polumarnh Be Find nolumarnhi ndam rana inan Murclantida install Infa More plugins at geneious com Installed Plugins Description 4 Collaboration Allows you to share your documents with other Complement or Reverse Only Allows Complementing or Reversing a Crispr Recognition Tool Wrapper A Geneious plugin wrapper for Bland DualBrothers Recombination Detection Detect recombination and EMBOSS Nucleotide Analysis Provides the EMBOSS Nucleotide Analysis Categories 1 Install plugin from a gplugin file Y Check for plugin updates now 4 Automatically check for updates to installed plugins Y Tell me when new plugins are released C Also check for beta releases of plugins Features The set of features availa
159. f Geneious Contact Support allows you to contact our Support team through Geneious Activate License lets you activate a license or connect to a license server Install FLEXnet installs the FLEXnet licensing service which is necessary to use FLEXnet licenses Borrow Floating License lets you borrow a license from a FLEXnet server if the main tainer of the server has provided you with a Borrow File Release Licenses releases any floating license you are currently holding and returns any local FLEXnet licenses to our server so they can be activated on a difference machine Buy Online sends you to our online store About Geneious gives details about the version of Geneious you are running and licens ing information Chapter 3 Importing and Exporting Data Geneious is able to import raw data from different applications and export the results in a range of formats If you are new to bioinformatics please take the time to familiarize yourself with this chapter as there are a number of formats to be aware of 3 1 Importing data from the hard drive to your Local folders To import files from local disks or network drives click File Import From file This will open up a file dialog Select one or more files and click Import If Geneious automatic file for mat detection fails select the file type you wish to import Figure 3 1 Files can also be dragged and dropped from your hard drive directly into Geneious and Geneious will automat
160. f a slightly offset version of this junction which allows this junction to be retained on the next pass e Reads using Indicates the number of reads that used this junction as part of their map ping during the second pass Junction Source amp Junction Destination Clickable links to the junction positions in the reference sequence When the destination is a different reference sequence this is prefixed with the sequence name followed by a colon e Color Annotations are colored from blue to green based on increasing values of Reads supporting discovery Reads spanning junctions may be represented in one of two possible ways For deletions under 1 000 bp the deletion is represented as a gap in the read For longer deletions or for structural variants two copies of the read appear in the contig where the fragment of the read extending past the junction is marked as trimmed 10 3 4 RNAseq mapping To map RNA sequence reads to a genome with introns choose Geneious for RNAseq as the Mapper in the Map to Reference setup dialog This function can map reads that span existing annotated introns or discover novel introns and fusion genes This function works in the same way as deletion and structural variant discovery section 10 3 3 for DNA mapping by analyzing how fragments of each read align to different regions of the reference sequence s and creating a junction annotation at the point where the read is split By default at least 2 re
161. f any read in a contig and the status bar will indicate the expect separation and expected separation between the reads 10 5 EDITING CONTIGS 143 10 5 Editing Contigs Editing a contig is exactly the same as editing an alignment in Geneious After selecting the contig click the Allow Editing button in the sequence viewer and you can modify insert and delete characters like in a standard text editor Editing of contigs is done to resolve conflicts between fragments before saving the final con sensus The normal procedure for this is to look through the disagreements in the contig as described above and change bases which you believe are bad calls to be the base which you believe is the correct call This is often decided by looking at the quality for each of the bases and choosing the higher quality one Geneious can do this automatically for you if you use the Highest Quality consensus Bases in the consensus sequence can also be edited which will update every sequence at the corresponding position to match what is set in the consensus You can also manually move a read mapped to a reference sequence to a specific position in the contig To do this select the read and right click then choose Move read to position and enter the position where you want the left most base in the read to sit D gt extract GRC mii Gf Allow Editing Add Edit Annotatior gt gt amp ue oO Consensus a A A Ce FWD 2
162. f it was deleted by mistake Pressing the Delete key is the easiest way to move the selected folder or documents to the Deleted Items folder To recover documents or folders from Deleted Items you can either drag and drop them to another folder or use Restore from Deleted Items Put Back from Deleted Items on Mac OS in the File menu to automatically move them to folder they were deleted from The Deleted Items folder should be cleared periodically to keep hard drive space free This can be done by selecting Erase All Deleted Items from the File menu Geneious will warn you if the Deleted Items folder contains a large amount of data To erase a document immediately without moving it to Deleted Items use Erase Document Permanently in the File menu or press Shift Delete Many of these actions can also be accessed by right clicking on a folder or document 4 2 Searching and filtering local documents The local database can be searched by clicking on the folder you wish to search then clicking the Search icon sesh in the top right of the toolbar Enter the desired term s in the text field and press enter or click the Search button Once a search starts the document table will initially be emptied then results will appear in the table as they are found see Figure 4 1 The Search button changes to a Cancel button while a search is in progress and this may be clicked at any time to terminate the search Feedback on a search progress is presente
163. f the applicable source annota tion Uncheck this option if you only want to return annotations which match across the full length of both sets Results A new annotation track will be created showing the results of the comparison The results panel in the compare annotations set up allows you to choose which comparison to return e A B C returns annotations found only in set A e B A C returns annotations found only in set B e C A B returns annotations found only in set C e A amp B amp C returns annotations common to all sets For a pairwise comparison when the annotation is common to both sets the result will have the name and type of annotation from set A but share properties from both sets if selected For a three way comparison properties from set A will be used More than one of these options can be selected at once either by checking the box next to the option or clicking the appropriate section of the venn diagram to the right Each result comparison is displayed on a separate track on the original sequence and a preview of these tracks is given in the Example panel Example 1 finding polymorphisms within a gene or feature e g CDS restriction site To return a track containing polymorphism annotations that are within another gene or fea ture such as a coding sequence select the Polymorphism annotation type for Set A and choose which track it is on or select anywhere if there are no tracks or you want to include polymor
164. ference Sequence project est Database of GenBank EMBL DDBJ sequences from EST Divisions est human Human subset of est est_mouse Mouse subset of est est_others Non Human non mouse subset of est gss Genome Survey Sequence includes single pass genomic data exon trapped sequences and Alu PCR sequences htgs Unfinished High Throughput Genomic Sequences phases 0 1 and 2 finished phase 3 HTG sequences are in nr pat Nucleotide sequences derived from the Patent division of GenBank PDB Sequences derived from the 3D structures of proteins from PDB month All new updated GenBank EMBL DDBJ PDB sequences released in the last 30 days RefSeq NCBI curated non redundant sets of sequences dbsts Database of GenBank EMBL DDBJ sequences from STS Divisions chromosome A database with complete genomes and chromosomes from the NCBI Reference Sequence project wes A database for whole genome shotgun sequence entries env_nt This contains DNA sequences from the environment i e all organisms put together Table 15 2 Protein BLAST databases Database Protein searches env_nr Translations of sequences in env_nt month All new updated GenBank coding region CDS translations PDB SwissProt PIR released in last 30 days nr All non redundant GenBank coding region CDS translations PDB SwissProt PIR PRF pat Protein sequences derived from the Patent division of GenBank PDB Sequences derived from 3D structure Brookhaven PDB RefSeq RefSeq protein sequences from NCBI s Referenc
165. ferential expression ratio it is useful to know whether or not that differential expression is statistically significant This is represented by a p value A num ber of advanced methods have been published for the calculation of p values based on a range of assumptions Many of these are compared by Soneson amp Delorenzi 2013 and they conclude that no single method is optimal under all circumstances and that very small samples sizes impose problems for all evaluated methods In this basic differential expression plugin in Geneious we have implemented a simple statisti cal test based on the assumption that the gene which each observed transcript came from is an independent event For a given gene the probability that a randomly selected transcript would come from that gene is calculated as number of transcripts mapped to that gene total number of transcripts from that sample This probability is normalized the mean probability between the two samples calculated and this mean un normalized for each sample This produces an expected prob ability that a randomly selected transcript from this sample comes from that gene assuming that this gene is not differentially expressed The Binomial Distribution is used to calculate the probability that an observed count at least as extreme as the observed one would be seen assuming this non differentially expressed mean probability The probabilities from each sample are multiplied together to form the p va
166. for download in PDF format it can also be stored in Geneious by saving it to your hard drive and then importing it This will allow full text searches to be performed on the article To view a pdf document either double click on the CHAPTER 3 IMPORTING AND EXPORTING DATA document in the Documents Table or click on the View Document button This opens the document in an external PDF viewer such as Adobe Acrobat Reader or Preview Mac OS X On Linux you can set an environmental variable named PDFViewer to the name of your external PDF viewer The default viewers on Linux are kpdf and evince GEE BibTex info gt y Estimating mutation parameters population history and genealogy simultaneously from temporally spaced sequence data Drummond AJ Nicholls GK Rodrigo AG amp Solomon W School of Biological Sciences University of Auckland 1001 Auckland New Zealand alexei drummond zoology oxford ac uk Genetics 2002 161 1307 20 Google Scholar Molecular sequences obtained at different sampling times from populations of rapidly evolving pathogens and from ancient subfossil and fossil sources are increasingly available with modern sequencing technology Here we present a Bayesian statistical inference approach to the joint estimation of mutation rate and population size that incorporates the uncertainty in the genealogy of such temporally spaced sequences by using Markov chain Monte Carlo MCMC integration The Kingm
167. forward strand CATGG C reverse strand c GGTAC 5 overhang 4 bases 3 overhang 4 bases Modify Vector Polylinker region to cut within 2 Bases 1 2 to 4 766 inclusive C Entire sequence Candidate Enzymes Enzymes annotated on insert Ncol 4 Enzyme set commonly used enzymes Cut vector with Ncol Product insert index 55 59 6 2 forward strand ye Ven A reverse strand GGTACC GGTACC vector index A ie ir oa _ Keep fragments which are not part of the product a coat GQ Figure 14 3 Restriction cloning options dialog 188 CHAPTER 14 CLONING or is blunt at both ends you can also choose to insert in both directions In this case two product documents will be created one for the insert in each direction The insert options also present a diagram showing the bases at each end of the insert fragment Vector Options Polylinker region to cut within These options let you choose what region within the vector sequence to look for enzymes to cut within Geneious will examine the vector sequence for enzymes that have cut sites within this region and none outside it You can specify the polylinker in the following ways Bases Used to explicitly specify the range of bases to use Entire sequence Used to specify that you can cut anywhere within the sequence e Candidate Enzymes These options let you choose which enzymes to look for on the vector sequence
168. from the Cloning menu The insert must be one of the following e A fragment which has already been digested This fragment cannot have any restriction site annotations on it The entire fragment will be inserted into the vector Overhangs will be taken into account e A sequence with two restriction site annotations The fragment resulting from digest ing this sequence and discarding the fragments from the ends will be inserted into the vector The vector must be a circular sequence You do not need to annotate the restriction sites used to cut the vector in advance the Restriction Cloning operation will do that for you This operation cannot deal with some aspects of molecular cloning such as triple ligation and the blunting or filling in of overhangs If you want to do a cloning operation outside the scope of this operation you will need to annotate restriction sites on the sequences involved digest the fragments modify them in the sequence viewer if necessary and then ligate them back together as a set of discrete steps Insert Options You cannot alter the insert used in the operation from the options but you can select what direction to insert in forward or reverse If the insert fragment has complementary overhangs 14 3 RESTRICTION CLONING 187 Insert A Overhangs are complementary so insertion can occur in both directions Insert forward only Insert reverse only _ Insert in both directions 55 59 6 2
169. from within Geneious by going to Help Online Resources Chapter 2 The Geneious main window Figure 2 1 shows the main Geneious window This has five important areas or panels a T E Back Forward Sequence Search Agents Align Assemble Tree Primers Cloning Detect Recomb ZAG XxX o 9 2 Toolbar Back Up Support Help Sources 1 of 8 selected w Local 0 EZ Name 4 Description v Sample Documents 0 COXII CDS Multiple alignment of 50 Cytochrome C Oxidase Subunit II genes Alignments 8 Z HIVenv 20 HIV Sete od Cloning 12 ig variable region in he ariable regions Contig Assembly 7 LysR family v Genomes 0 Pairwise protein Document ta bi kiwifruit and papaya Bacteria 3 People bm fictitious characters Eukaryotes 157 Three Kingdoms Multiple alignment of Alanyl tRNA synthetase from organims of three di Human 25 Transcript variants Multiple alignment of 4 variants of MAPK10 genes Plants 42 By Viruses 6 Shared Databases Operations Y NCBI Gene B Genome 2 Nucleotide 2 PopSet Protein PubMed snp Structure Taxonomy 3 UniProt 887 Using 179 3996 MB memory Y Hide ISE Annotations Virtual Gel Distances Text View Info BD Cr Extract GRC 6 Translate di Add Edit Annotation gf Allow Editing gt 63 E 1 10 20 30 40 50 Consensus Identity Orangutan Gorilla
170. geneioug Geneious 9 0 Biomatters Ltd December 10 2015 Contents 1 Getting Started 1 1 Downloading amp Installing Geneious 2 i244 bed corista LE SCO EO 2 ae eS BSS AE Ga Se y See eS 13 o o e he Eee ee ee Peewee A L4 Licensing oe Ee es ae ie ak BN SS PERE SG OS SESS a GS Loe 7 Ces rae eos ee ere se II 2 The Geneious main window 21 TheSources Panel oe ie Gee eh RARA ROE Bw A ARA 22 The Document ISE lt cedi ced ramea ee b ee AE HARE RS 23 The Document Viewer Panel 64 55 68 808 8 4983 ee4ee4 be 5 ees Oe The Rep Panel sore Reba oe ERA RES ae ES HE EEE ES EES 25 The Toolbar oda BES REDEE oe A Eee 26 Weneious menu bar ophions ys cora RARA ASA 3 Importing and Exporting Data 3 1 Importing data from the hard drive to your Local folders de Dia mpu Irma ak ed cw KE PEE FRE ee eK CORRE EEE RY 3 3 Importing files from public databases ooo oo oo ooo A O AS PoR ok a ES EES ED a ED RRS BAS DRE oe EXI Mes ras Oe Se a OS EE EPEC REESE ERS 14 15 16 17 18 18 20 21 22 23 CONTENTS 36 Pinte and Saving mee o AAA A AA 45 Managing your Local Documents 47 4 1 Organizing your local documents lt lt cs sd ss vi 47 4 2 Searching and filtering local documents o o ooo ooo ooo 49 A A II HHS EEE ERO 53 4A Batch A IS DESH ew OE Ew ee OS SR 54 45 Backing up your local doctiments lt s ss oresar do dede as 54 260 Document History es eo ar AA Oe p Ow ie Be 56 C
171. gins installed Click the question mark button next to the method to see a list of the advantages and disad vantages of each assembler The sensitivity setting specifies a trade off between the time it takes to assemble and the accuracy of the assembly Higher sensitivity is likely to result in more reads being assembled 10 2 DENOVO ASSEMBLY 133 C Assemble by lst part of name separated by Hyphen _ Assemble each sequence list separately O Use 100 E of data Suitable for genome size between 11 KB and 45 KB Method Assembler Geneious Sensitivity Medium Sensitivity Fast O Memory Required Between 85 MB and 91 MB of 916 MB Note Paired reads can be set up or changed using Sequence gt Set Paired Reads Trim Before Assembly Results Assembly Name ReadeNamelAsiemby E Use existing trim regions C Save assembly report O Remove existing trim regions from sequences _ Save list of unused reads O Trim sequences Options C Save in sub folder M Save contigs M Maximum 8 Save consensus sequences 2 veranos Gre Gy 2 Do not trim Figure 10 4 Basic de novo assembly options 134 CHAPTER 10 ASSEMBLY AND MAPPING e Trim Sequences Select how to trim the ends of the sequences being assembled See section 10 1 1 e Results Allow you to choose an assembly name and what to return in your results By default only the assembled c
172. gion All of these are expressed in base pairs from the beginning of the sequence and are as follows Included Region Specifies the region of the sequence within which primers are allowed to fall This must surround the target region and allows you to choose a small region on either side of the target in which primers must lie e Target Region Specifies which region of the sequence you wish to amplify and unless the advanced options allow otherwise the forward and reverse primers must fall somewhere 13 1 DESIGN NEW PRIMERS 167 outside this region e Product Size Specifies the range of sizes which the product of a primer pair can have The product size is the distance in bp between the beginning of the forward primer to the end of the reverse primer e Optimal Product Size Specifies the preferred size of the product Setting this will mean primer pairs that have a product size close to this will be chosen over those that do not Warning Setting these options can cause the primer design process to take considerably longer to complete The final option in this section is Number of Pairs to Generate which specifies how many candidate pairs of primers and DNA probes to generate and is compulsory Setting this to 1 will give you only the primer pair which was considered best by the set parameters 13 1 2 Cloning primers This option allows you to design primers to amplify a specific region Only the included region can be set and the
173. gle folder In this view each hit sequence in each alignment is annotated with a Search hit annotation If you mouse over the annotation you can bring up the values for E value pair wise identity Grade etc To display these values in a table switch to the Annotations tab in the sequence viewer and add these columns to the table by clicking the Columns button 15 3 NCBI BLAST Geneious is able to BLAST to many different databases held at NCBI These databases are listed in the Tables 15 1 and 15 2 and can be selected in the Databases drop down menu in the BLAST set up dialog You must be able to connect to the internet from within Geneious to BLAST to NCBI and if you are behind a proxy server you may need to enter your proxy server settings under Tools Preferences Connection Settings as described in Section 1 2 5 If you have a mirror of the NCBI BLAST databases you can set Geneious to use this by going to Tools Add Remove Databases Set Up BLAST Services This will bring up a dialog that allows you to change the setup for various search services in Geneious Choose NCBI using the service drop down box at the top of the dialog Enter the URL for the mirror and click OK to apply the new settings 206 CHAPTER 15 BLAST Table 15 1 Nucleotide BLAST databases Database Nucleotide searches nr All non redundant GenBank EMBL DDBJ PDB sequences no EST STS GSS or HTGS sequences genome Genomic entries from NCBI s Re
174. gn a partner primer to match an existing one for example a reverse primer for a forward or vice versa It also allows you to design a probe to match a pair of primers If any documents were selected which either are primer sequences or contain primer annota 165 166 CHAPTER 13 PCR PRIMERS 8 0 0 Design New Primers Select Task 4 Design New F Design with Existing Please cite Primer3 if you publish result md Forward Primer DNA Probe M Reverse Primer Task gt Included Region vie Ye Target Region _ Product Size Between Optimal Product Size Number of pairs to generate 1 z gt Tm Calculation gt Characteristics Advanced x _Cancel Hoe A Figure 13 1 The primer design dialog tions then these will be made available for selection as primers in a drop down box Selected sequences are treated as primer or probe sequences if they are 150bp in length or less For each of these options Generic or Cloning primers can be designed 13 1 1 Generic primers This option will design standard PCR primers according to the region input options you select These options allow you to specify what part of a sequence you wish to amplify Most options are optional and can be enabled or disabled with the associated check boxes beside them If you have selected a region in the sequence before opening the primer dialog then this region will automatically be used for Included Region and Target Re
175. gnment is not displayed make sure the alignment is selected in the Document Table and that the Document Viewer is set to Alignment View There is also an option to perform the alignment using ClustalW or MUSCLE two widely used alignment programs or via a translation alignment where DNA sequences are aligned by their Figure 2 4 The Help Panel 2 5 The Toolbar The toolbar contains several icons that provide shortcuts to common functions in Geneious including BLAST Sequence Search Agents that search databases for new content even while you sleep Align Assemble Tree building Primer Design and Help The Back and Forward options help you move between previous views in Geneious and are analogous to the back and forward buttons in a web browser The V option shows a list of previous views The other features that can be accessed from the toolbar are described in later sections 2 6 GENEIOUS MENU BAR OPTIONS gt Back Forward Sequence Search Agents Sources v Local 0 v Sample Documents 0 3D Structures 5 Alignments 7 P Contig Assembly 6 Genomes 4 Linnaeus Blast 1 Nucleotide Documents Plasmids from NEB 27 Protein Documents 5 Restriction Enzymes 2 Tree Documents 4 B Deleted Items 0 Searches 0 B Server Databases EB Operations Collaboration Y NCBI 7 Gene B Genome 2 Nucleotide gt DonSor A Align Assemble Tree Primers Cloning
176. h any existing Jabber service such as Google Talk but we recom mend using the Geneious default talk geneious com You can even access several Jabber accounts at the same time which is particularly convenient if you wish to set up and run your own Jabber server section 19 5 3 This chapter shows you how to e Create a new collaboration account e Search for and add contacts to your account Share local folders with your contacts e Search your contacts as you would an online database e Set up and run your own Jabber server 221 222 CHAPTER 19 COLLABORATION 19 1 Managing Your Accounts When the Collaboration plugin is installed you will see the empty Collaboration service in the Sources Panel and the Collaboration submenu under Tools You can open the Add New Ac count dialog by either right clicking Ctrl click on Mac OS X on Collaboration in the Sources Panel and clicking Add New Account in the popup menu or by selecting the same option from the Collaboration submenu 19 1 1 Add New Account In this dialog you are given the options of creating a new account on the server or entering the details for an existing account e g if you want to access an account from an additional computer If you choose to create a new account Geneious will attempt to automatically register your account on the server at the end of this process 7 Add New Account 5 Create a new account on the server This account already exists j
177. h improves the accuracy of consensus and variant calling For more information click the help question mark button next to the fine tuning options in the Map to Reference setup dialog If you just wish to use a reference sequence to help construction of the contig where the reads extend beyond the length of the reference then you have two options With iterative fine tuning reads can extend a bit further past the ends of the reference sequence on each iteration so make sure you set the number of iterations high enough Or you could select all sequences including the reference and use the De Novo assembler 10 3 3 Deletion and structural variant discovery DNA mapping Geneious can discover structural rearrangements and arbitrarily large deletions from paired or unpaired reads by analyzing how fragments of each read align to different regions of the reference sequence s To enable this option check Find structural variants and deletions of any size If you only want to find deletions up to a specified size check Find large deletions up to For this operation Geneious makes two passes during mapping On the first pass each read mapped will generate candidate junctions sites for structural variants based on where frag ments of the read align to different regions of the reference sequence s The more reads that support a candidate junction the more likely it will be used during the second pass The second pass involves mapping reads using
178. he main page Geneious will follow all hyperlinks between the pages and external hyperlinks beginning with http will be opened in the user s browser If you want to include figures and diagrams in the pages just put the image files in the same folder and reference them with lt img gt tags like a normal HTML document supported image formats are GIF JPG and PNG If you want to include Geneious documents in your tutorial simply place them in the same folder as the html documents and they will automatically be imported into Geneious with the tutorial If you want to link to them from the tutorial pages create a hyperlink pointing to the file in the HTML document For example to create a link to the file sequence fasta in your tutorial folder use the HTML lt a href sequence fasta gt click here lt a gt To open more than one document from a link separate the filenames with the pipe character for example lt a href sequenc fastalsequence2 fasta gt click here lt a gt Note that geneious files must contain only one document to be imported automatically with the tutorial You can add a short one line summary by writing your summary in a file called summary txt case sensitive and putting it in the tutorial folder Make sure that the entire summary is on the first line of the file as all other lines will be ignored 217 218 CHAPTER 17 GENEIOUS EDUCATION Once you have all your files together put the contents
179. he standard 84 CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES 000 Edit Meta data Types Existing Types Primer Info Name Source Description Depth Decimal gt Constraints LatLong Text gt Constraints Date Collected Date gt Constraints Text Constraints True False _ i Whole Number Decimal Date Drop down list d Create Delete Cancel fox Figure 5 9 The Edit Meta Data Types window search fields These are listed under the Advanced Search options in the Document Table see section 4 2 1 From then on you can use them to search your Local Documents If you have more than one Field in a meta data type they will all appear as searchable fields in the search criteria Filtering Meta data values can be used to filter the documents being viewed To do so type a value into the Filter Box in the right hand side of the Toolbar see section 4 2 4 Only matching documents will be shown Sorting Any meta data fields added to documents will also appear as columns in the Docu ment Table These new columns can be used to order the table Chapter 6 Parent Descendant tracking Many documents in Geneious are the output of an operation run on a set of input documents The input documents of the operation are known as the parents of the output and the output documents the descendants or children of the input Those parent documents may them selves be the descend
180. her documents Duplicates can be identified by sequence name database ID e g accession or by the residues bases and the Search Scope can be set so that it checks within either a selected set of documents all documents in a folder or in the sequences of a single alignment or sequence list When searching for duplicates within sequences of a single alignment or sequence list two options are available for displaying results once the search has run e Select earlier duplicates in list This will select all but one copy of a duplicated docu ment allowing the duplicates to easily be deleted or moved to another folder leaving one copy behind e Extract unique sequences Unique sequences will be extracted to a new sequence list and the sequence names modified to show the duplicate count for that sequence For 58 CHAPTER 4 MANAGING YOUR LOCAL DOCUMENTS large data sets or removing duplicates in paired reads or removing non exact duplicates see Remove Duplicate Reads using BBTools If you are searching for duplicates within a folder or multiple select documents you can choose to select either the most recently or least recently modified copy Remove Duplicate Reads For identifying non exact duplicates removing exact duplicates from large data sets or remov ing duplicates on paired read data sets use Remove Duplicate Reads from the Sequence menu This tool runs Dedupe from the BBTools suite For a detailed explanation of any Ded
181. hes go from right to left rather than left to right e Transform branches allows the branches to be equal like a cladogram or proportional Leaving it unselected leaves the tree in its original form e Ordering orders branches in increasing or decreasing order of length but within each clade or cluster Show root branch displays the position of the root of the tree has no effect in the un rooted layout e Line weight can be increased or decreased to change the thickness of the lines represent ing the branches e Show selected subtree only shows only the part of the tree that is selected or the entire tree if there is no selection 162 CHAPTER 12 BUILDING PHYLOGENETIC TREES 12 5 5 Show Tips Node and Branch Labels If you are unfamiliar with tree structures please refer to Figure 12 1 for a diagram of tips nodes and labels Show tip labels This refers to labels on the tips of the branches of the tree Tip labels can be any of the fields on your document and can be set in the Display option To select multiple fields to display at the tips hold down the command control key while selecting Show node labels This refers to labels on the internal nodes of the tree If you are viewing a consensus tree you can display consensus support here or you can display the node heights Show branch labels This refers to labels the branches of the tree You can display substitu tions per site branch lengths here or for a c
182. hich comprise this meta data type This includes custom meta data types that you have created and saved in the past When you re ready hit OK to begin importing When Geneious is done you may be presented with the option of grouping the sequences you imported into a sequence list This option is recommended if you re importing very large sets of sequences 13 4 Primer Database The Primer Database consists of all the oligonucleotide documents that exist in your Local or Shared Databases The oligonucleotide Y document type is a short nucleotide sequence representing either a primer or a probe The text view lists the primer characteristics Im GC etc These properties can be shown in the document table Tm is shown by default but you can turn on others by right clicking on the table header Oligo sequences are created via one of the following methods e Extract a primer probe annotation from a sequence e Select Sequence New Sequence from the menu and choose Primer or Probe as the type of the new sequence e Select one or more existing primer sequences maybe ones imported from a file then click Primers Convert to Oligo to transform them into oligo type sequences e Import primer sequences from a comma separated file csv and choose Primer or Probe as the sequence type see section 13 3 If you select a target sequence and go to Test with Saved Primers or Design Primers De sign With Existing Geneious will find all oligo
183. hronized storage location without any problems Folders in a Shared Database will show up under the Shared Databases icon in the Sources panel once the user has logged into the database A Shared Database can be used for everything a local database is used for This includes col laboration Take note that unread status agents and shared folders belong to individual users rather than the database For example Bob may see a document as unread but Joe will see that same document as read if he has read it 20 1 Supported Database Systems To use a database as a Shared Database Geneious requires that it support transactions with an isolation level set to SERIALIZABLE Supported databases systems include Microsoft SQL Server PostgreSQL Oracle and MySQL It is possible to use other database systems if you provide the database driver see section 20 2 1 Shared Databases have been tested using e Microsoft SOL Server 2008 R2 e PostgreSQL 8 4 e Oracle 11g Express Edition e MySQL 5 1 229 230 CHAPTER 20 SHARED DATABASES 20 2 Setting up After a database is set up correctly multiple users can connect to it and use it as their storage location just as if they were using their own local database Follow these steps to set up your database for use with Geneious e Install a supported database management system if you do not already have one e Create a new database with your desired name Make sure that you have a user that has rights t
184. ically de termine the file type The different file formats that Geneious can import are described in detail in the next section Files imported from disk are imported directly into the currently selected local folder within Geneious If no folder is selected Geneious will open a dialog which lets you specify a folder File name Import Cancel Files of type 3 0 structure documents pdb mol cml gpr hin nwo Is 3 D structure documents pdb mol cml pr hin nwo ace PHRAP file Format ace ace 1 txt Chromatogram ab abi ab1 scf Clustal aln DNA Strider str DNAStar seq pro Endnote 8 0 or 9 0 xml Fasta Autodetect Fasta Fas fa mpfa fna fsa txt Figure 3 1 File import options 33 34 CHAPTER 3 IMPORTING AND EXPORTING DATA In Geneious R9 and above it is possible to import an entire folder and all its subfolders and files into Geneious in one step To do this choose File Import From Directory If the folder has subfolders the folder structure will be retained when it is imported into Geneious 3 2 Data input formats Geneious version 9 0 can import the following file formats Format Extensions Data types Common sources BED bed Annotations UCSC Common Assembly Format caf Contigs Sequencher Clustal aln Alignments ClustalX CSFASTA osfasta Color space FASTA ABI SOLID DNA
185. identify documents in the document table matching chosen keywords It is located in the top right hand corner of the Main Toolbar Type in the text you are searching for and Geneious will display all the documents that match this text and hide all other documents in the Document Table To view all the documents in a folder clear the Filter box of text or click the button It is also possible to sort sequences in a given folder by their similarity To use this function select a single sequence in the document table and right click then choose Sort Sort by simi larity will rank all other sequences by their similarity to the selected sequence The most similar sequence is placed at the top and the least similar sequence at the bottom This also produces an E value column describing how similar the sequences are to the selected one The Remove Sort by Similarity option will remove the E value column and return the table to its previous sorting Filtering on the fly Filtering can be used while searching for documents via public databases filtering data as it is being downloaded Type in the appropriate text in the Filter Box and only those documents that match both the original criteria as specified by the search terms and the Filter text will be displayed This is an effective way of filtering within your search results 4 3 Find Duplicates Find Duplicates under the Edit menu is used to identify duplicate copies of sequences and ot
186. if any If the insert fragment was produced from a sequence with two restriction site annotations the fragments on either side of the restriction site annotations will also be kept 14 4 GIBSON ASSEMBLY 189 14 4 Gibson Assembly The operation will generate sequences with compatible overlaps that can ligate to each other after a partial chew back with a T5 exonuclease The overlaps are created by extension overlap PCR the corresponding primers will automatically be generated and displayed in a report document and as annotations on the resulting sequences Figure 14 4 below shows the Gibson assembly options e0 Gibson Assembly Backbone Digestion of Cloning vector lt Choose ert each fragment separately Assemble fragments end on end Drag to set order 5 Promoter 3 DCN gene 3 Min Overlap Length 18 bp Min Overlap Tm 48 C Y Save in sub folder Gibson Assembly y Tm Calculation y Formula Breslauer et a Salt correction SantaLucia 1 Concentration Settings Monovalent 50 mM Oligo 50 5 nM Divalent 1 5 4 mM dNTPs 0 6 mM Me Cancel ok Figure 14 4 Gibson Assembly options dialog with extended options showing e From Geneious 8 1 onwards it is not necessary to select the insert and vector prior to opening the Gibson Assembly options If you select only a single sequence prior to se lecting Gibson Assembly then this sequence will appear as the insert
187. igher than the 60 threshold to call an A With the G included the total quality is 30 25 30 85 which is higher than the 60 threshold so a consensus call of R will be made The quality assigned to this R will be the sum of the bases that agree with the consensus call minus the bases that disagree which is 30 25 30 15 70 For alignments or contigs with a reference sequence the If no coverage call setting can be used to control what character the consensus sequence should use when the reference sequence has no coverage Options available are X N or Ref A represents an unknown character potentially a gap If Ref is selected then the consensus is assigned whatever character the reference sequence has at that position Note that if any sequence in the alignment contig has an internal gap in it that is still considered valid coverage at that position and this setting will not apply Choose Call N if Quality below to change consensus bases to N s if the quality is below the threshold that you set This is particularly useful for exporting sequences to file formats which do not preserve quality for example FASTA To work with the consensus sequence in a downstream analysis you must first Extract it from your alignment To do this click on Consensus to select the entire sequence then click Extract to extract it to a new sequence document Alternatively go to Tools Generate Consensus Sequence This operation
188. imming e Enable per folder document binning When turned on the Set Binning Parameters option is added to the Sequence menu This allows you to select an individual folder or set of documents and set the binning parameters to use on those documents instead of the global ones set in the Preferences 1 2 GENEIOUS SETUP 13 1 2 2 Choosing where to store your data Geneious stores your data in a folder called Geneious X Y Data where X and Y are the ver sion of Geneious you are using which is stored separately from the application itself When Geneious first starts up you will be asked to choose a location for this folder The default lo cation in the user s home directory is normally the best option Although it s possible to store your data on a network or USB drive so you can access it from other computers this is not rec ommended because it can have adverse effects on performance Please do not use a DropBox folder to store your data This may corrupt your data To store your data somewhere different to the default simply click the Select button in the welcome window and choose an empty folder on your drive where you would like to store your data To change the location of your Geneious database at a later date go to Tools Preferences General The Data Storage Location shows the current location of your database see Figure 1 1 Click the Browse button to select a new location Geneious will offer to either copy your exi
189. imum Likelihood and Bayesian MCMC exter nal plugins for specialist software are available see section 12 3 4 for a full list These can be downloaded from the plugins page on our website or within Geneious by going toPlugins under the Tools menu 12 1 Phylogenetic tree representation A phylogenetic tree describes the evolutionary relationships amongst a set of sequences They have a few commonly associated terms that are depicted in Figure 12 1 and are described below Branch length A measure of the amount of divergence between two nodes in the tree Branch lengths are usually expressed in units of substitutions per site of the sequence alignment Nodes or internal nodes of a tree represent the inferred common ancestors of the sequences that are grouped under them Tips or leaves of a tree represent the sequences used to construct the tree Taxonomic units These can be species genes or individuals associated with the tips of the tree A phylogenetic tree can be rooted or unrooted A rooted tree consists of a root or the common ancestor for all the taxonomic units of the tree An unrooted tree is one that does not show the position of the root An unrooted tree can be rooted by adding an outgroup a species that is distantly related to all the taxonomic units in the tree 153 154 CHAPTER 12 BUILDING PHYLOGENETIC TREES Root Branch Node Tip Figure 12 1 Phylogenetic tree terms For information on viewing and formatting tr
190. imum and maximum of the lengths of the sequences Confidence mean When viewing sequences containing quality scores e g chromatograms or NGS reads this gives the mean of the confidence scores for the currently selected base calls Confidence scores are provided by the base calling program not Geneious and give a measure of quality higher means a base call is more likely to be correct An untrimmed value is also displayed if the selected region contains trims 5 3 EDITING SEQUENCES 75 e Expected Errors When viewing sequences containing quality scores this gives the approx imate number of errors that are statistically expected in the currently selected region This is calculated by converting the confidence score for each base call to the error probabil ity using the formula 109 10 For example a base with a quality score of 30 will have an error probability of 0 001 The expected errors value is then calculated by summing up the error rates for each base This also has a value for the untrimmed selection if the region contains trims GC content For documents that are created or modified in Geneious 8 1 or later the GC content can also be viewed in the GC column in the document table The GC column shows the percentage of A C G T U S W nucleotides that are either G C or S Ambiguous bases that contain a mixture of GC and non GC bases e g R Y M K are excluded from the calculation This field is available on all nuc
191. ins menu where you can install or uninstall plugins e Preferences see section 1 2 1 Many plugin options will also appear in this menu when installed such as Classify Sequences Sequence Classifier plugin Submit to Genbank Genbank submission plugin and Collabo ration see chapter 19 CHAPTER 2 THE GENEIOUS MAIN WINDOW 2 6 5 Sequence Menu This contains several operations for manipulating nucleotide and protein sequences including processing NGS reads prior to assembly New Sequence Create a new nucleotide or protein sequence including oligos from residues that you can paste or type in See section 5 1 Extract Region Extract the selected part of a sequence or alignment into a new document Reverse Complement Reverse sequence direction and replace each base by its comple ment See section 5 4 Translate Creates a new protein document from the translated DNA see section 5 5 Back Translate Creates nucleotide version of the selected protein document see section D92 Circular Sequences Sets whether the currently selected sequences are circular This ef fects the way the sequence view displays them as well as how certain operations deal with the sequences eg digestion See section 5 2 3 Free End Gaps Alignment Sets whether the currently selected alignment has free end gaps This effects calculation of the consensus sequences and statistics Change Residue Numbering Changes the original residue numbering
192. ion Geneious also has the option of refining the multiple sequence alignment once it is done Refining an alignment in volves removing sequences from the alignment one at a time and then realigning the removed sequence to a profile of the remaining sequences The number of times each sequence is re aligned is determined by the refinement iterations option in the multiple alignment window The resulting alignment is placed in the folder containing the original sequences A profile is a matrix of numbers representing the proportion of symbols nucleotide or amino acid at each position in an alignment This can then be pairwise aligned to another sequence or alignment profile When pairwise aligning profiles mismatch costs are weighted propor tional to the fraction of mismatching bases and gap introduction and gap extension costs are proportionally reduced at sites where the other profile contains some gaps In some cases building a guide tree can take a long time since it requires making a pairwise alignment between each pair of sequences The build guide tree via alignment option may speed this part by taking a different route First make a progressive multiple alignment using a random ordering and use that alignment to build the guide tree Notice that while this usually speeds up the process it may not if the sequences are very distant genetically 800 Alignment Geneious Alignment MUSCLE Alignment Translation Align Clust
193. ion phylogenetic or mutation studies These alignments usually describe evolution and population variation The PopSet database contains both nucleotide and protein sequence 3 3 IMPORTING FILES FROM PUBLIC DATABASES 43 data and can be used to analyze the evolutionary relatedness of a population The Entrez Protein database This database contains sequence data from the translated coding regions from DNA sequences in GenBank EMBL and DDBJ as well as protein sequences sub mitted to the Protein Information Resource PIR SWISS PROT Protein Research Foundation PRF and Protein Data Bank PDB sequences from solved structures The PubMed database This is a service of the U S National Library of Medicine that includes over 16 million citations from MEDLINE and other life science journals This archive of biomed ical articles dates back to the 1950s PubMed includes links to full text articles and other related resources with the exception of those journals that need licenses to access their most recent issues Entrez SNP In collaboration with the National Human Genome Research Institute The Na tional Center for Biotechnology Information has established the dbSNP database to serve as a central repository for both single base nucleotide subsitutions and short deletion and insertion polymorphisms The Entrez Structure database This is NCBI s structure database and is also called MMDB Molecular Modeling Database It contains three di
194. ion on how this is calculated There is a Base Call Quality color scheme which is selected by default for alignments of all chromatograms This assigns a shade of blue to each base based on its quality Dark blue for confidence lt 20 blue for 20 40 and light blue for gt 40 The consensus is also colored with this scheme where the confidence of a given base in the consensus is equal to the maximum confidence from the bases at that site in the alignment There is a Mapping Quality color scheme for reads mapped to a reference sequence A mapping quality represents the confidence that the read has been mapped to the correct location For a read with mapping quality Q the probability that it has been incorrectly mapped is 109 10 For example a read with a mapping quality score of 20 has a 1 chance of having been incorrectly mapped Reads that could be mapped to multiple locations will have a maximum mapping quality score of 3 which indicates it had at least a 50 probability of mapping elsewhere Mapping qualities have a maximum value of 254 for consistency with the SAM BAM format If a sequence has no mapping quality i e the document was produced in a version of Geneious prior to 8 1 or imported from a SAM BAM file that didn t have mapping quality then it will be colored gray Mapping quality for the sequence under the mouse is also displayed in the status bar All mappers use heuristics to calculate mapping qualities For unpaired reads the Ge
195. ions Settings Change proxy Lan Settings settings Figure 1 3 Checking browser settings If you have downloaded a plugin file from our website or obtained one from another source in gplugin format you can install it by clicking Install plugin from a gplugin file and brows ing to the location of the file You can also install plugins by dragging the gplugin file into Geneious If you are running Geneious as an administrator then any plugin you install will be installed for all users on the same computer If you are not running as an administrator then plugins will only be installed for the current user account When upgrading plugins Geneious may display a message indicating that Geneious needs to be restarted in order to complete the plu gin upgrade If Geneious was being run as an administrator it needs to be restarted as an administrator to complete the plugin upgrade Note that on Windows you cannot drag and drop plugins in to Geneious while running as an administrator Installed Plugins lists all the plugins you current have installed Click the uninstall button next to a plugin to remove it Other options for managing plugins are e Check for plugin updates now Checks if there are any new versions available for the plugins you have installed e Automatically check for updates to installed plugins If checked Geneious will check for new versions of your installed plugins each time the program is started e Tell me when n
196. ires that pair of reads on each end of the contig match and that those reads don t intersect with each other anywhere else in the contig i e pairwise contigs will never circularise A circular contig will contain reads at either end marked with arrows which denotes that these reads span the origin and link back around to the other end of the assembly The consensus se quence produced from this contig will also be circular The Topology column in the Document table lists whether a given contig is circular or linear 10 2 1 The de novo assembly algorithm The sequence assembler in Geneious is flexible enough to handle read errors consisting of either incorrect bases or short indels It can handle data from any type of sequencing machine with reads of any length including paired reads and mixtures of reads from different sequencing machines hybrid assemblies The de novo assembly algorithm used is a greedy algorithm which is similar to that used in multiple sequence alignment 1 For each sequence a blast like algorithm is used to find the closest matching sequence among all other sequences 2 The highest scoring sequence and its closest matching sequence are merged together into a contig reverse complementing if necessary This process is repeated appending se quences to contigs and joining contigs where necessary 3 For paired read de novo assembly 2 sequences with similar expected mate distances are given a higher matching sco
197. is split into seven main menus File Edit View Tools Sequence Annotate amp Predict and Help 28 CHAPTER 2 THE GENEIOUS MAIN WINDOW 2 6 1 File Menu This contains standard options for managing files including import export see chapter 3 deleting saving and backing up files see 4 5 creating renaming and moving folders It also contains options for printing and saving image files see 3 6 2 2 6 2 Edit Menu This menu contains the standard editing functions for transferring information from within documents to other locations both within and outside of Geneious Cut Copy Paste Delete and Select All This menu also contains options for finding and renaming documents and their contents e Find in Document can be used to find text or numbers in a selected document see section 423 e Find Next and Find Previous finds the next or previous match for the text you specified in the Find in Document dialog e Find Duplicates see section 4 3 e Batch Rename see section 4 4 e Go to base residue see section 5 2 2 2 6 3 View Menu This contains several options and commands for changing the way you view data in Geneious Back Forwards and History allow you to return to documents you had selected previ ously e Search is discussed in section 4 2 e Agents are discussed in section 3 4 e Next unread document selects the next document in the current folder which is unread Table Columns contains the same functio
198. isn t valid for the latest version Downgrading requires that the new version of Geneious is uninstalled first to avoid there be ing vestiges of the old copy in place Once this is done the old version can be reinstalled and Geneious will start up and see the old data folder For instructions on how to import data from a newer version into an old version see the section below on File compatibility 1 3 2 File compatibility Geneious data files are backwards compatible to version 6 0 Thus files that were created in version 6 1 or higher can be exported in geneious format and opened in any version back to 6 0 If you are using an earlier version than version 6 0 you won t be able to open files in geneious format that were created in a newer version Entire Geneious databases are not backwards compatible so when you upgrade you should accept the offer to keep a backup of your existing database If you then need to downgrade to an earlier version you can swap back to your old database and if you are on Geneious 6 0 or above export any files you changed in the new version in geneious format and import them into your old database If you wish to export data back into a version earlier than 6 0 you will need to export the files in a common format such as fasta or genbank 1 4 LICENSING 19 1 4 Licensing Licenses can be managed under the Help menu in Geneious using the following options 1 41 Activate License In this window you can activat
199. kflow go to Manage Workflows under the Workflows menu Here you have the option to create a New Workflow View Edit an existing Workflow or Copy and edit an existing Workflow Each of these options opens an Edit Workflow window See example in Figure 16 2 where you can name your Workflow describe it s function specify an icon for display in the Workflows menu share via a Shared database and build edit your Workflow using the Add Step Delete Step buttons Each Workflow is made up of one or more Steps A Step may be an Operation for example perform Muscle alignment or a special Step for example Group Documents Each Step accepts one or more documents as input and produces one or more documents as output which are then used as input to the next step in the Workflow All documents selected when the Workflow is run are provided as input to the first Step unless specified otherwise see section 16 2 2 for further information The output from the final Step of a Workflow is saved in your Geneious database Outputs from intermediate steps are not saved unless you include the Save Documents Branch option after the Step A document is a single entry that can be 16 2 CREATING AND EDITING WORKFLOWS 213 Workflow Name Align DNA twith MAFFT then build tree with Mr Bayes lt html gt lt center gt Builds a DNA alignment using MAFFT and then builds a tree from that Description alignment lt center gt lt html gt Icon gt Choose
200. l other sequences are inserts e If any complications occur eg when more than one TOPO site is detected or when a linear sequence with TOPO site is selected it will print a message in this box also showing how the corresponding sequence is processed if the user clicks OK e The resulting sequences will be optionally saved in a sub folder 14 8 CRISPR site finder The CRISPR Cas9 system is an RNA guided endonuclease technology for gene editing This system requires guide RNA gRNA comprised of a 20 bp target sequence next to a PAM Pro tospacer Adjacent Motif to direct the Cas9 enzyme to the cleavage site The Find CRISPR Sites 196 CHAPTER 14 CLONING TOPO Cloning _ TA Cloning Blunt Cloning 9 Directional with overlap CACC Vectors Vector A Vector B All other sequences are treated as inserts and will be inserted into each vector V Save in sub folder TOPO Results Figure 14 6 TOPO Cloning options dialog tool will search for gRNA CRISPR sites in your selected sequence and can score them based on on target activity or potential off target interactions To search for CRISPR sites first select the sequence you want to target This can be a full se quence document or a selection in a sequence For best performance the search region should be limited to 1000 bp Open the Find CRISPR Sites tool in the Cloning menu Check Any where in sequence or Selected region depending on if y
201. ld guide tree via alignment faster Refinement Options Refinement iterations 2 8 C Create an alignment without actually aligning the sequences 1 A Fewer Options Cancel o Figure 21 3 Log in to Geneious Server M Delete Started T 23 Nov 2011 1 29 PM 4 22 Nov 2011 4 26 PM Finished 22 Nov 2011 4 25 PM Finished 22 Nov 2011 3 40 PM Finished 22 Nov 2011 3 26 PM Finished 21 Nov 2011 9 40 AM Finished Paired Reads assem Search DCN gene Progress Running Run Location Geneious Server Local Local Local Geneious Server Local unning Pop Out Select results Select results Download Results Figure 21 4 Operations table showing Geneious Server and local jobs 21 4 GENEIOUS SERVER ENABLED PLUGINS 237 21 4 Geneious Server enabled plugins This table details plugins which work with Geneious Server Note that some of these plugins only run on Geneious Server and will not run on the local desktop implementation of Geneious Plugin Local Server Geneious Alignment Yes Yes MUSCLE Alignment Yes Yes ClustalW Alignment Yes Yes Realign Region Yes Yes Translation Align Yes Yes MAFFT Alignment Yes Yes Consensus Align Yes Yes Profile Align Yes Yes Mauve Genome Yes Yes LASTZ Alignment Yes Yes Geneious Tree Builder Yes Yes Consensus Tree Builder Yes Yes MrBayes Yes Yes PHYML Yes Yes P
202. le restriction enzymes If you have created your own restriction enzyme set from your local database then this will also be listed see below for how to create such a document e Enzymes must match X to Y times only returns restriction enzymes which cut the se quence X to Y times Results for enzymes that cut the sequence more or less than this will be discarded If you set X to be 0 when this operation is complete it will report which candidate enzymes do not cut the sequence The restriction enzyme information included in Geneious was obtained from Rebase available for free at http rebase neb com Like many restriction enzymes EcoRI is methylation dependent and cuts only if the second A in the recognition sequence is not methylated to N6 methyladenosine 14 2 DIGEST INTO FRAGMENTS 183 e Specifying cut regions To specify a region of sequence where you want the enzyme to cut or not cut choose one of the options below and use the base counters to specify a sequence range that the options apply to If you have selected a region of sequence in the sequence viewer clicking the refresh arrow next to the base number counters will copy the selected region to this setting The following options are available Cut Anywhere Returns enzymes which cut anywhere in the entire sequence It is not possible to select a subregion with this setting Must only cut between Returns enzymes which only cut between the specified bases
203. lected folder or folders presented in table form A local folder may contain any mixture of documents such as DNA sequences protein sequences journal articles sequence alignments and trees Figure 2 2 If you cannot see all of the columns in the document table you may want to close the help panel to make more room For information on how to search and filter documents in the Document Table see section 4 2 Selecting a document in the Document Table will display its details in the Document View Panel Selecting multiple documents will show a view of all the selected documents if they are of similar types e g selecting two sequences will show both of them in the sequence view The easiest way to select multiple documents is by clicking on the checkboxes down the left hand side of the table Standard keyboard controls can also be used Shift and Ctrl command click 2 2 THE DOCUMENT TABLE 23 Name Xidentical Journal Title First Author PMID Sequence Resi URLFR Summary 4 Avirus reveals population str aes Area e g STEE ci ace ido Science Roman Biek 16439664 http ww i 22 Population genetic estimation POPulation genetic estimation of the loss of genetic diversity BMC Evol Biol Charles TT 16556318 http w during horizontal transmission of HIV 1 Relaxed phylogenetics and dating with confidence Aa Relaxed phylogenetics and da Alexei J Drummond Simon Y W Ho Matthew J Phillips Andrew PLoS Biol A
204. leotide sequences contigs align ments and sequence lists that were created or had their sequences last modified in Geneious 8 1 or later For contigs and alignments the consensus sequence and reference sequence if any are excluded from the calculation For sequences within an alignment contig or list the GC column only shows the overall value for the alignment To see a table of GC percentages for all individual sequences within an alignment or contig the sequences need to be extracted to stand alone sequences Alterna tively individual values can be viewed in the statistics panel by clicking on the name of the sequence to select it Sequences in a list or alignment can be sorted by GC content by right clicking in the sequence viewer and choosing Sort GC 5 3 Editing sequences To edit sequence s or an alignment click the Allow Editing toolbar button You can manually enter or delete sequence or use any of the standard editing operations such as Copy Ctrl command C Cut Ctrl command X Paste Ctrl command V Paste With out Annotations Shift Ctrl command V Paste Reverse Complement and Undo Ctrl command 2Z All operations are under the main Edit menu or can be accessed by right clicking in the se quence view and selecting the option from the popup window To insert sequence click at the position where you want to insert the sequence and type or paste it in Normally existing residues will be shifted to the
205. lete genome Figure 4 1 Searching the Document Table Wild card searches When you are looking for all matches to a partial word use the asterisk Asterisks can be placed at the beginning end or in the middle of a word For example typing oxi would return matches such as oxidase oxidation oxido reductase and oxide Searching for CO I would return matches for COI and COXI Similarly you can use a question mark to represent any single character This feature is available only for local documents and not NCBI or UniProt searches 4 2 1 Advanced Search options To access advanced search click the More Options button inside the basic search panel To return to basic search click the Fewer Options button Switching between advanced and basic will not clear the search results table The advanced search allows you to search for specific terms in specific fields of your docu ments The fields available for a search can be found in the left most drop down box all fields potentially available on your local documents are listed here If you have defined a new type of meta data in Geneious and that meta data field has been added to a document then this field will also be available to search Advanced Search also provides you with a number of options for restricting the search on a field depending on the field you are searching against For example if you are using numbers to search for Sequence length or No of no
206. lexei J Dru 16683862 http bic MA modified cc_cd11_M13F_C05 modified cc_cd11_M13F_C05_022 ab1 Length 597 GCTCACGA MA cc_cd11_M13F_C05_022 ab1 cc_cd11_M13F_C05_022 ab1 Length 597 gctsacgatgc LWA modified cc_cd12_M13F_DOS modified cc_cd12_M13F_D05_021 ab1 Length 618 GCTSCGATG A Nucleotide alignment 6 Alignment of 2 sequences cc_cd11_M13F_C05_022 ab1 82 8 E New nucleotide sequence New nucleotied sequence A new nucleotide sequence entered ACGATCAC K 1996YangGeneticsv144p194 1996YangGeneticsv144p1941 pdf 3 tree txt tree txt 4 tips a fs E FE tree3 txt tree3 txt 1 Trees Figure 2 2 The document table when browsing the local folders Double clicking a document in the Document Table displays the same view in a separate win dow To view the functions available for any particular document or group of documents right click Ctrl click on Mac OS X on a selection of them These options vary depending on the type of document Document Table features Editing Values can be typed into the columns of the table This is a useful way of editing the information in a document To edit a particular value first click on the document and then click on the column which you want to edit Enter the appropriate new information and press enter Certain columns cannot be edited however eg the NCBI accession number Copying Col
207. lign A ClustalW Alignment Profile Aligr Cost Matrix 65 similarity 5 0 4 0 Gap open penalty 12 Gap extension penalty 311 7 Alignment type Global alignment with free end gaps Automatically determine sequences direction Y More Options Cancel Co Figure 9 2 Options for nucleotide pairwise alignment The score of a pairwise alignment is matchCount x matchCost mismatchCount x mismatchCost For each gap of length n a score of gapOpenPenalty n 1 x gapExtensionPenalty is subtracted from this Where e gapOpenPenalty The gap open penalty setting in Geneious e gapExtensionPenalty The gap extension penalty setting in Geneious e matchCost The first number in the Geneious cost matrix e mismatchCost The second number in the Geneious cost matrix e matchCount The number of matching residues in the alignment e mismatchCount The number of mismatched residues in the alignment When doing a Global alignment with free end gaps gaps at either end of the alignment are not penalized when determining the optimal alignment This is especially useful if you are aligning sequence fragments that overlap slightly in their starting and ending positions e g when using two slightly different primer pairs to extract related sequence fragments from different samples You can also do a Local Alignment if you want to allow free end overlaps rather than just free end gaps in one alignment
208. ligo from the popup menu that appears If you select just two sequences you have the additional option of determining their pair characteristics Determining the pair characteristics of two primer sequences can be used to see if two sequences can pair and how well they do so Note Sequences longer than 36 bp can still be converted into oligo sequences but their charac teristics cannot be calculated as this is the size limit that Primer3 sets for accurate Tm calcula tions 13 8 PRIMER EXTENSIONS 177 13 8 Primer Extensions You can add a primer extension to an existing oligonucleotide Y sequence by selecting Primers Add 5 Extension You can add your own sequence a restriction site and or Gateway cloning site Multiple extensions can be added in one go and the preview window in the 5 extension dialog box shows how these extensions will be arranged on the primer The order of 5 additions can be edited by dragging and dropping them in this window Primer extensions can also be added at the time the primer is designed from within the Design New Primers setup options e 0 Add 5 Extension Add Restriction Site Gateway Site Rag G Binding regio 3 Cancel ok Figure 13 7 Adding 5 extensions to a primer Once added the 5 extension is shown in bold on the primer sequence and is not covered by the primer annotation as shown in Figure 13 8 These extensions will not change the binding region of the prime
209. lly performed before assembling a contig This is because the noise introduced by low quality regions and vector contamination can pro duce incorrect assemblies To trim vectors primers and poor quality bases using the Geneious tools select the sequences you wish to trim and choose Annotate and Predict Trim Ends This option can also be performed at the assembly step by checking the trim sequences option in the assembly set up Geneious R9 also provides a plugin for trimming using the BBDuk algorithm from the BBTools suite This is can be installed by going to Tools Plugins Trim Ends can soft or hard trim your sequences If you wish to soft trim choose to Annotate 125 126 CHAPTER 10 ASSEMBLY AND MAPPING new trimmed regions in the Trim Ends set up The trimmed sequence will then remain vis ible but will be annotated with Trimmed annotations Sequence annotated with a trimmed annotation is ignored by the assembler when constructing a contig and will not be included in the consensus sequence calculation So although the trimmed regions are visible they do not affect the results of assembly at all Soft trims can be adjusted as needed or deleted completely Dragging the ends of the trim annotation will make the newly untrimmed sequence visible and part of the consensus Figure 10 1 If you wish to remove the trimmed sequence completely hard trim choose Remove new trimmed regions from sequences 450 460 469 477 4
210. lowing e Next search in x time e g 18 hours The agent is waiting until its next scheduled search and it will search when this time is reached e Searching These are shown in bold The agent is currently searching e Disabled The agent will not perform any searches e Service unavailable The agent cannot find the database it is scheduled to search This will happen if the database plugin has been uninstalled or if for example the Collabora tion contact is offline currently e No search scheduled The agent is enabled but doesn t have a search scheduled To correct this click the Run now button in the agent dialog to have it search immediately and schedule a new search Deliver To This names the destination folder for the downloaded documents This is usually your Local Documents or one of your local folders Note If you close Geneious while an agent is running it will stop in mid search It will resume searching when Geneious is restarted 3 4 3 Manipulating an agent Once an agent has been set up it can be disabled enabled edited deleted and run All these options are available from within the Agents dialog e Enable or disable an agent by clicking the check box in the Enable column e Run Now Cause the agent to search immediately e Cancel If the agent is currently searching click this to stop the search e Edit Click this to change an agent s database search criteria destination or
211. lt and or shift key while clicking the plus or minus button zooms in to fit the selected region in the available viewing area P zooms to 100 The 100 zoom level allows for comfortable reading of the sequence Ra zooms out so as to fit the entire sequence in the available viewing area Zooming can also be quickly achieved by holding down the zoom modifier key which is the Ctrl key on Windows Linux or the Alt Option key on Mac OS X and clicking as described below When the zoom key is pressed a magnifying glass mouse cursor will be displayed e Hold the zoom key and left click on the sequence to zoom in e Hold the zoom key and Shift key to zoom out e Hold the zoom key and turn the scroll wheel on your mouse if you have one to zoom in 64 CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES and out e Hold the zoom key and click on an annotation to zoom to that annotation You can also pan in the Sequence View by holding Ctrl Alt command Alt on Mac OS X and clicking on the sequence and dragging 5 2 2 Selecting part of a sequence Within the sequence viewer there are several ways to select part of a sequence or select a subset of sequences from a list alignment or assembly e Mouse dragging Click and hold down the left mouse button at the start position and drag to the end position By using the Ctrl Windows Linux or command Mac keys it is possible to select multiple regions of a sequence or alignment as d
212. lue Results Results are displayed as an additional Expression level annotation track on the reference se quence Each annotation produced will have the following properties e Differential Expression p value A p value indicating the confidence that this gene is differentially expressed 152 CHAPTER 11 ANALYSIS OF ASSEMBLIES AND ALIGNMENTS Differential Expression Absolute Confidence The negative base 10 log of the p value This field is useful for filtering on For example in the Filter box on the results you could type absolute confidence gt 6 without quotes to only show genes that we are fairly confident have differential expression Since filtering works on partial property names in most cases just a shorter filter like abs gt 6 is sufficient unless your genes have existing numeric properties containing the text abs Differential Expression Confidence The negative base 10 log of the p value but ad justed to be negative for genes that are under expressed in sample 2 compared to sample 1 or positive for over expressed genes The results are colored based on this property from blue for under expressed genes through to white for genes that are not differen tially expressed through to red for genes that are over expressed Confidence coloring reaches maximum intensity at 8 Differential Expression Ratio The ratio of the normalized values between the two sam ples but ratios less than one are replaced with
213. ly best scoring matches are saved although there is an option to map the read to all best scoring locations Paired reads are given an additional score penalty based on their distance from their expected distance so that they prefer mapping close to their expected distance with as few mismatches as possible but they can also map any distance apart if an ideal location is not found The final optional fine tuning step at the end shuffles the gaps around so that they reads better align to each other rather than the reference sequence For details on how mapping qualities are calculated see See section 10 4 For further details and for a comparison of the Geneious reference assembler to other software see the Geneious Mapper white paper 10 4 Viewing Contigs Contigs in Geneious are viewed and edited in exactly the same way as alignments There are several features in the sequence viewer which are worth taking special note of when viewing contigs e The consensus sequence is normally of particular interest and this is always displayed at the top of the sequence view labeled Consensus 10 4 VIEWING CONTIGS 141 When all sequences in a contig or alignment have quality information attached then you can select the Highest Quality consensus type This almost removes the need for manually editing the contig because this consensus chooses the base with the highest total quality at each position See section 9 4 for more informat
214. ly link source and extracted documents checkbox in the relevant dialog see Figure 6 2 otherwise they will be created with permanently inactive links Biret Es Extraction name A Aa os F Actively link source and extracted documents ox cancel Figure 6 2 Extract dialog with active link checkbox 6 1 EDITING LINKED DOCUMENTS 87 6 1 Editing Linked Documents When you make changes to a document that is the parent of another document you will be given the opportunity to either propagate the changes deactivate the link which can later be reactivated see Lineage View Section 6 2 or save the changed document as a new copy Figure 6 3 You may also simply back out of this process by choosing to cancel which will return you to your unsaved changes Note that if you choose to deactivate the link this dialog will not be displayed upon subsequent saves of the parent document unless the link is reactivated again at some future time a Actively Linked Descendants nese This document has actively linked descendants View Descendants What would you like to do Propagate changes and update descendants Deactivate links to descendants so they are not updated Save as copy without descendants save _ canc Figure 6 3 Actively Linked Descendants dialog In order to aid with your decision making the dialog allows you to view the document s de scendants in a smaller cut down version of the Lineage
215. m62 score matrix has a value of 2 for K vs R 5 2 THE SEQUENCE VIEWER 69 If you raised the threshold to 3 then this column would no longer be considered 100 similar If the column consisted of 9 K s and 1 R then continuing with the threshold value of 3 the 9 K s which make up 90 of the column would now be colored the dark grey 80 100 range while the single R would remain uncolored If instead the column consisted of 7 K s and 3 R s still with threshold 3 then 70 of the column is now similar so those 7 K s would be colored the lighter grey 60 80 range Alternatively going back to the default threshold value of 1 and with a column consisting of 7 K s 2 R s and 1 Y now since the 7 K s and 2 R s have similarity exceeding the threshold whereas the Y is not that similar to K and R the K s and R s will be colored dark grey since they make up 90 of the column Hydrophobicity color scheme This colors amino acids from red through to blue according to their hydrophobicity value where red is the most hydrophobic and blue is the most hydrophilic The values the color scale is based on are given in Figure 5 4 These values are taken from http biochem ncsu edu faculty mattos CrystallographyTutorial AminoAcids htm Polarity color scheme This colors amino acids according to their polarity as follows Yellow Non polar G A V L I F W M P Green Polar uncharged S T C Y N Q Red Polar
216. mensional biomolecular experimentally or programmatically determined structures obtained from the Protein Data Bank Entrez Taxonomy This database contains the names of all organisms that are represented in the NCBI genetic database Each organism must be represented by at least one nucleotide or protein sequence 3 3 3 Literature searching Geneious allows you to search for relevant literature in NCBI s PubMed database The results of this search are summarized in columns in the Document Table and include the PubMed ID PMID first and last authors URL if available and the name of the Journal When a document is selected the abstract of the article is displayed in the Document Viewer Text View tab along with a link to the full text of the document if available and a link to Google Scholar both below the author s name s Figure 3 2 As well as the abstract and links Geneious also shows the summary of the journal article in BibTex format in a separate tab of the Document Viewer BibTex tab BibTex is the standard ISTgX bibliography reference and publication management data format and the information in the BibTex screen can be imported directly into a TEX document when creating a bibliography Alternatively a set of articles in Geneious can be directly exported to an EndNote 8 0 compati ble format This is usually done when creating a bibliography for Microsoft Word documents Note If the full text of the article is available
217. n interactive 3D view of the protein structure including support for displaying the protein s secondary structure when the appropriate information is available PDF PDF stands for Portable Document Format and is developed and distributed by Adobe Sys tems It contains the entire description of a document including text fonts graphics colors links and images The advantage of PDF files is that they look the same regardless of the software used to create them Some word processors are able to export a document into PDF format Alternatively Adobe Writer can be used You can use Geneious to read store and open PDF files 3 2 DATA INPUT FORMATS 39 ACE PHRAP assembly Ace is the format used by the Phrap Consed package created by the University of Washington Genome Center This package is used mainly to assemble sequences GCG PileUp alignment The PileUp format is used by the pileup program a part of the Genetics Computer Group GCG Wisconsin Package PIR NBRF sequences Format used by the Protein Information Resource a database established by the National Biomedical Research Foundation Qual quality Phred scores Quality file which must be in the same folder as the sequence file FASTA format for the quality scores to be used Unformatted sequence A file containing only a sequence RSF rich sequences RSF Rich Sequence Format files contain one or more sequences that may or may not be related In addition to the
218. nality as the popup menu for the document table header See section 2 2 for more details e Open document in new window Opens a new window with a view of the currently selected document s 2 6 GENEIOUS MENU BAR OPTIONS 29 e Expand document view expands the document viewer panel in the main window out to fill the entire main window Selecting this again to return to normal e Split Viewer Left Right creates a second copy of the document viewer with the two views laid out side by side Split Viewer Top Bottom creates a second copy of the document viewer with one on top of the other Document Windows Lists the currently open document windows Selecting one from this menu will bring that document window to the front 2 6 4 Tools Menu e Align Assemble see section 9 2 and section 10 respectively e Tree see section 12 e Primers see section 13 e Cloning see section 14 BLAST Perform a BLAST search such as NCBI Blast to find sequences that are similar to the currently selected sequence s See section 15 e Add Remove Databases options for setting up and configuring NCBI and custom BLAST see section 15 4 3 e Extract Annotations see section 8 1 4 Strip Alignment Columns see section 9 3 4 e Concatenate Sequences or Alignments see section 5 3 1 e Generate Consensus Sequence see section 9 4 e Workflows access built in Workflows and create new ones see chapter 16 e Plugins Takes you to the Plug
219. nd multiple alignment al gorithms are available The consensus sequence used for each alignment is a 100 consensus with gaps ignored 9 3 Alignment viewing and editing Alignments are displayed in the viewer below the document table in the same way as indi vidual sequences See section 5 2 for details on the sequence viewer including basic controls such as zooming in and out wrapping sequences setting colors and selecting individual or multiple sequences from an alignment For a description of alignment statistics available in the 9 3 ALIGNMENT VIEWING AND EDITING 119 8090 Pairwise Multiple Align A Geneious Alignment MUSCLE Alignment ClustalW Alignment ealign Rec Translation Align Genetic code Standard a Translation frame 1 M Treat first codon as start of coding region Protein alignment options Geneious Alignment Cost Matrix Blosum62 Gap open penalty 12 Gap extension penalty 311 Alignment type Global alignment with free end gaps Y More Options Cancel ok Figure 9 4 Options for nucleotide translation alignment Statistics tab see section 5 2 9 To edit an alignment you must first click the Allow Editing button on the toolbar above the sequence viewer Alignments are edited in the same way as for individual sequences for details on editing operations and shortcut keys see section 5 3 If the consensus sequence of an alignment or assembly is edited the changes are applied to all se
220. nd or modify it If you don t drag the documents from a database search into your local folders the results will be lost when Geneious is closed For more information on how to move files between folders in your database see 4 1 1 Note When searching the Genome Gene or PopSet databases the documents returned are only summaries To download the whole genome select the summary s of the genome s you would like to download and the click the Download button inside the document view or just above it Alternatively you can choose Download Documents in the File menu and in the popup menu when document summary is right clicked Ctrl click on Mac OS X The size of these files is not displayed in the Documents Table Be aware that whole genomes can be very large and can take a long time to download You can cancel the download of document summaries by selecting Cancel Downloads from any of the locations mentioned above 42 CHAPTER 3 IMPORTING AND EXPORTING DATA 3 3 1 UniProt This database is a comprehensive catalogue of protein data It includes protein sequences and functions from Swiss Prot TrEMBL and PIR 3 3 2 NCBI Entrez databases NCBI was established in 1988 as a public resource for information on molecular biology Geneious allows you to directly download information from nine important NCBI databases and perform NCBI BLAST searches Table 3 1 Table 3 1 NCBI databases accessible via Geneious Database Coverage Gene G
221. ndant relationships manage links and navigate between documents Figure 6 6 Sequence View Annotations Dotplot Self Virtual Gel DNA Fold _ Enzymes Fragments Text View Ma by i oO Properties M Show Operations M Show Inactive Links Goto r Export History Lineage Parents Descendants O TetR repressiple GFP generator inserted into pSB1C3 O TetR repressiple GFP generator inserted into pSB1C3 v Restriction Cloning Today at 3 43 PM v B Extract PCR Product Today at 3 45 PM O psB1C3 E Terr repressiple GFP generator inserted into pSB1C3 PCR Produc E Terr repressiple GFP generator Legend Selected Document Active Link Inactive Link Figure 6 6 The Lineage View All active links appear as green text whilst inactive links appear as black text and the docu ment currently being viewed and which is the root of the parents tree and the descendents tree appears in blue Each s document s name is displayed along with an icon similar to the document table denoting what type of sequence it is Also displayed in the viewer are the operations that generated each set of children along with 6 2 THE LINEAGE VIEW 89 the time at which the operation was run and the type of operation If preferred these operations can be hidden by unchecking the Show Operations checkbox providing a layout which is akin to Vector NTI You can also choose to view only inactive links by unchecking the Show Inactive Links checkbox This will hi
222. ne If your contact is online they will appear blue A contact online in Geneious will have the orange Geneious G behind them A contact online in some other program like a chat client will have a speech bubble behind them 19 2 2 Rename Contact This option allows you to change the name that you know another contact by This is the name the contact will appear under in the contact list and in chats it is only visible to you Rename Contact 9 Name for blah blah Geneious User Figure 19 5 Rename Contact dialog box 226 CHAPTER 19 COLLABORATION 19 2 3 Remove Contact If you no longer wish to share documents with a contact you can remove that contact by right clicking Ctrl click on Mac OS X the contact in the Sources panel and selecting Remove Con tact This deletes you from their contact list as well If you find that a contact has disappeared from your list this may be the reason 19 3 Sharing Documents Select one of your local folders Select Share Folder from the File menu Alternatively right click Ctrl click on Mac OS X on a local folder and select the same option e If you share a folder all documents in that folder are shared e If you share a folder all sub folders of that folder are shared e If you share a folder it is available to all your contacts 19 4 Browsing Searching and Viewing Shared Documents Folders that your contacts have shared will appear beneath that contact just as they
223. ne containing queries which get a hit in the database the other containing queries which don t Details about the hits and alignments are discarded This can be used to filter contamination eg human from sequencing reads You can also specify how much of each matching sequence to retrieve from your database e Matching region Just the region of the database sequence which matches the query e Matching region with annotations The region of the database sequence which matches the query plus any annotations on that sequence e Extended region with annotations The matching region plus additional flanking regions upstream and downstream e Full sequence with annotations The entire database sequence this could be large and slow Geneious also allows you to specify most of the advanced options that are available in BLAST To access the advanced options click the More Options button which is in the bottom left of the BLAST options The available options vary depending on the kind of BLAST search you have selected For details on each of the options you can hover your mouse over the option to see a short description or refer to the BLAST documentation from NCBI 15 2 BLAST results Once a search has started a results subfolder will be created in the same folder as your query sequence Search progress is shown in the document table The search can be cancelled by clicking on the red square labelled Stop 15 2 1 BLAST hit table If y
224. neious mapper assigns a mapping quality of 20 the number of additional mismatches in the second best location the read maps to For paired reads the individual unpaired mapping qualities are calculated but these are increased by up to 20 depending on how close the best pair is to the expected insert distance compared with the second best pair e The sequence logo graph has an option to Weight by quality This is very useful for identifying low quality regions and resolving conflicts Finding regions of low high coverage In addition to the coverage graph which gives you a quick overview of coverage under the Annotate Predict toolbar is the Find Low High Coverage feature This feature annotates all regions of low high coverage which you can then navigate through using the little left and right arrows next to the coverage annotations in the controls on the right You can set the threshold low high coverage by either specifying an absolute number of sequences or a number of standard deviations from the mean coverage The find low high coverage tool can also be used to record the minimum mean and maximum coverage of each annotation of a particular type on the reference sequence To do this in the Only Find In section of the options turn on Annotations in reference sequence of type and choose Create annotations of same type on reference sequence 142 CHAPTER 10 ASSEMBLY AND MAPPING ce Extract GF R C E Translate Allow Editing gt
225. ng largely continuous diagonal indicates that the sequences are related along their entire length e Sequences with some limited regions of similarity will display short stretches of diagonal lines Diagonals on either side of the main diagonal indicate repeat regions caused by duplica tion e A random scattering of dots reflects a lack of significant similarity These dots are caused by short sub sequences that match by chance alone For more information on dotplots refer to the paper by Maizel and Lenk 1981 9 2 SEQUENCE ALIGNMENTS 111 9 2 Sequence Alignments Over evolutionary time related DNA or amino acid sequences diverge through the accumula tion of mutation events such as nucleotide or amino acid substitutions insertions and deletions A sequence alignment is an attempt to determine regions of homology in a set of sequences It consists of a table with one sequence per row and with each column containing homologous residues from the different sequences e g residues that are thought to have evolved from a common ancestral nucleotide amino acid If it is thought that the ancestral nucleotide amino acid got lost on the evolutionary path to one descendant sequence this sequence will show a special gap character in that alignment column 9 2 1 Pairwise sequence alignments There are two types of pairwise alignments local and global alignments A local alignment is an alignment of two sub regions of a pair of s
226. ng the down arrow will select the sequences in batches of 10 Go to position To jump to a particular base in a sequence you can use amp Go to base under the Edit menu for amino acid sequences this appears as Go to Residue This allows for the instant navigation to a particular nucleotide or amino acid coordinate for any sequence in the current document selection It also allows the selection of a particular region of sequence either for individual sequences or across sequence lists and alignments or the selection of particular sequences out of a sequence list Formatting examples are given in the setup dialog Go to Position also appears next to the sequence viewer when in genome view see section 5 2 4 5 2 3 Circular sequences When a circular sequence is selected the default view is to display the sequence as circular The view can be rotated by using the scrollbar at the bottom or by turning the mouse wheel Even though a sequence is circular you can display it as a linear sequence using the Linear view on circular sequence checkbox under the Layout section of Advanced To change a linear sequence into a circular sequence select the sequence then go to Sequence Circular Sequence then click Save This will join the ends of your sequence up to create a circular sequence but does not check for overlapping ends Circularization will affect how some operations such as restriction digest and map to reference deal with the sequen
227. nts in a format specified by an older version of Geneious if that version is still supported In most cases this will allow for users using Geneious 8 1 or later to seamlessly share documents with users of any version back to Geneious 6 0 However be aware that if the document format has changed since the compatibility version then users of the newer version may find their documents missing properties or bug fixes that have been added since the format changed This setting will apply to all users using the database with Geneious 8 1 or higher and can only be set by a user with an Admin role for the Everybody group You will find this setting by right clicking on the root folder and choosing Administration and then Set Document Com patibility Version Chapter 21 Geneious Server 21 1 Introduction to Geneious Server If your site has a Geneious Server installed you can use it to offload many of the tasks that Geneious would normally run locally on to the server taking the processing load off your own computer Once a job is sent to Geneious Server it will either be processed on the server itself a so called standalone installation or be handed off to a cluster running Oracle Grid Engine LSF or PBS schedulers To use Geneious Server a server side user account is required The server side user account will have a server access license associated with it Alternatively your server may have a queue licensing system which allows a certain numbe
228. o create tables e Use the Connect to a database button to connect to your database If the database has not been set up usually the case if you are following these instructions Geneious will detect this and set up the database This will only succeed if you have permission to create tables on the database e Make sure any other users of the database have SELECT INSERT UPDATE and DELETE rights otherwise they will not be able to use the Shared Database as intended There are two ways you can use your database with multiple users The simple way is just to use the Shared Database as a shared local database If this is all you want then you are now done with setup Alternatively you may want to restrict access to particular folders with groups and roles To do this please refer to section 20 4 1 Your database should now be ready to use with Geneious Now all users can connect to the database by clicking on Shared Databases in the Sources panel and then clicking Connect to a setup database This will bring up a dialog for the user to enter in the database details 20 2 1 Supplying your own Database Driver Shared Databases were designed with the supported databases in mind and packaged with database drivers for them However Geneious allows you to supply your own jdbc database driver if you want to You may want to do this because you have an updated driver or because you have a driver for an unsupported database It is not guaranteed that
229. obabilities and thus may have a negative base score E g For Q10 P error 0 1 For Q30 P error 0 001 10 1 READ PROCESSING 129 So with an error probability cutoff of 0 05 a base with Q10 has a base score of 0 05 0 1 0 05 and a base with Q30 would have a base score of 0 05 0 001 0 049 The trimming algorithm then calculates the running sum of the base score across the sequence If the sum drops below zero it is set to zero The part of the sequence not trimmed is the region between the first positive value of the running sum and the highest value of the running sum i e the highest scoring segment of the sequence Everything before and after this region is trimmed 10 1 2 Removing duplicate reads To remove duplicate reads from NGS datasets use Remove Duplicate Reads under the Se quence menu This function runs Dedupe and will remove duplicate sequences that are either exact matches subsequences or sequences within some percent identity It can also find over lapping sequences and group them into clusters For a detailed explanation of any Dedupe setting hover the mouse over the setting or click the help question mark button next to the custom options under More Options 10 1 3 Error correction and normalization of reads Prior to de novo assembly it can sometimes be useful to error correct the data or to normalize coverage by discarding reads in regions of high coverage This functionality is available using Error Cor
230. obe is available if a DNA Probe is being designed or tested These two sections are quite similar the DNA probe section has a subset of the options avail able in the primer section This is because primers are usually chosen in pairs and so several options can be set for how pairs are chosen y Characteristics DNA Probe Size Min 18 Optimal 20 7 Max 27 Tm Min 57 Optimal 60 gt Max 6311 GC Min 20 Optimal 50 Max 80 Product Tm Min 0 Optimal 0 gt Max oj Max Tm Difference 100 GC Clamp olls Max Dimer Tm 47 Max Poly X Silt Max 3 Stability 911 _ Allow primers inside target with penalty _ Primer Picking Weights _ Allow Degeneracy Figure 13 2 Primer characteristics options 13 1 5 Primer Picking Weights At the bottom of the Characteristics panel there is a Primer Picking Weights button Clicking this brings up a second dialog containing many more options The purpose of all of these options is to allow you to assign penalty weights to each of the parameters you can set in the options The weight specified here determines how much of a penalty primers and probes get when they do not match the optimal options The higher the value the less likely a primer or probe will be chosen if it does not meet the optimal value 13 1 DESIGN NEW PRIMERS 169 Some of the weights allow you to specify a Less Than and Greater Than This is for options which allow you
231. of the folder in a zip file with the exten sion tutorial zip Subfolders within the zip file are supported in Geneious R8 and higher 17 2 Answering a tutorial Import the tutorial document into Geneious use File Import From file or drag it in The tutorial document and any associated geneious documents will be imported into the currently selected folder The tutorial itself will be displayed in the help pane on the right hand side of the Geneious window If you accidentally close the help pane you can display it by choosing Help from the Help menu If the tutorial requires you to enter answers click the edit button at the top of the tutorial window and type your answer in to the space provided Click the Save button when you are done If the tutorial has a link to a Geneious document when you click the link the document will be opened in the document viewer Any changes you make to this document will be preserved when you export the tutorial When you have finished the tutorial export it by selecting the tutorial document and choosing File Export Selected Documents from the main menu Make sure that Geneious Tutorial File is selected as the filetype and then give it a name and click Export Chapter 18 Saving operation settings option profiles Profiles allow you to save the settings for almost any analysis operation in Geneious so they can be loaded later or shared with others Eg the recommended trimming parame
232. omatogram traces sequence alignments or assemblies and includes graphs for GC content Identity Cov erage and Quality The graphs available for display depend on the type of sequence you are viewing More detail on graphs for nucleotide and protein sequences is given in section 5 2 7 and for alignment and contig graphs is given in section 9 3 2 gt Annotations On sequences containing annotations this tab will show a yellow arrow It contains con trols for turning on and off annotations of each type customising the way each type is displayed and filtering based on annotation name or type See chapter 8 for more infor mation on working with annotations ay Live Annotate and Predict Contains real time annotation generators such as Annotate from Database Find ORFs and Transfer Annotations To use one of these turn on the check box at the top of the 68 CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES generator you want to use and annotations will immediately be added to the sequence You can then change settings for the generator and the annotations will change on the sequence in real time as you do If you want to save the annotations permanently on the sequence click Apply Restriction Analysis This behaves similarly to the Live Annotate amp Predict section above Please refer to chap ter 14 for full details Sw Advanced Contains advanced options for controlling the look of sequences and alignments includ ing wrap
233. omes chromosome DNA Sequence Tagged Sites dbsts GenBank EMBL DDBJ STS Divisions DNA Metagenomic Proteins env_nr Proteins from WGS metagenomic projects AA Environmental samples env_nt DNA Expressed sequence tags est GenBank EMBL DDB EST Divisions DNA EST Human Human expressed sequence tags DNA EST Mouse Mouse expressed sequence tags DNA EST Others Non Mouse non Human expressed sequence tags DNA Genomic Survey Sequences gss DNA High Throughput Genomic Sequences htgs DNA Month New or revised in the last 30 days AA or DNA nr GenBank EMBL DDB PDB RefSeg or GenBank PDB SwissProt PIR PRF AA or DNA Patented Protein Sequences pat From Patent division of GenBank MA or DNA Protein Data Bank pdb Sequences derived from PDB structures AA or DNA NCBI Reference Genomic Sequences refseq_genomic DNA Reference Proteins refseq_protein NCBI Protein Reference Sequences AA UniProtKB Swiss Prot Non redundant UniProtKB SwissProt sequences AA Whole Genome Shotgun Contigs wgs DNA Maximum Hits E more onions Figure 15 6 Searching a Custom BLAST database Chapter 16 Workflows Workflows allow you to group Operations together to reduce the number of steps required to perform an often used combination of Operations Options for each Operation may be precon figured or some or all options exposed for configuration when the Workflow is run Geneious provides a n
234. on this can be time consuming Also note that reactivating will cause any unsaved changes to any direct or indirect descendants to be overwritten since this involves a complete recompute from the parent documents You will be warned about this before Geneious allows you to reactivate Finally you may export the currently selected document highlighted in blue in the view di rectly via the Export button Doing so will bring up a dialog Figure 6 8 From here you can choose to export parents or descendants only or both as well as choose to export only those documents that are actively linked in the hierarchy Similarly to how unchecking the Show In active Links checkbox works unchecking Inactively linked documents here will mean that the export will stop as soon as it finds in inactively linked parent or descendant depending on the relevant direction and stop exporting down that branch of the lineage 90 CHAPTER 6 PARENT DESCENDANT TRACKING Exporting Human proinsulin modified inserted into Cloning vector pEF F3 EGFP F3 mcs Also indude Y Descendants Parents Y Inactively linked documents Reset to Defaults Figure 6 8 Export Dialog Chapter 7 RNA DNA and Protein structure viewer 7 1 RNA DNA secondary structure fold viewer This viewer will appear when the selected nucleotide sequence is less than 3000bp long If the sequence is DNA the tab will be labelled DNA Fold and if it is RN
235. onsensus or bootstrapped tree you can display consensus support or bootstrap support For node and branch labels the font can be set using the Font Size options in the tab The tree viewer will shrink the font size of some labels if they cannot all fit in the available space The lower end of the range specifies the minimum size that the tree viewer is allowed to shrink the label font to The font sizes for the tip labels are set using the Font button in the toolbar above the tree viewer Significant Digits sets how many digits to display if the value the node is displaying is numeric 12 5 6 Automatically collapse subtrees This option is available in version 9 onwards and enables groups of similar nodes to be col lapsed into a single node that represents that subtree The maximum distance within the sub trees is determined by the Subtree Distance slider Use this option to help navigate trees with many nodes and tips Collapsed nodes are labeled with the name of one of the tips a count of how many tips the subtree contains and the maximum distance between the top of the subtree and any of the tips within it Double clicking a node in a tree will force it to expand or contract Automatically Collapse Subtrees will not override this state To reset the state of double clicked nodes in the tree click Reset state of X nodes X is the number of nodes with a manually expanded or collapsed state 12 5 7 Show scale bar This displays a scale
236. ontigs are saved but you can also choose to return an as sembly report lists of used or unused reads and the consensus sequences The assembly report summarises the assembly statistics and lists which fragments were successfully assembled and which contig they went in to along with a list of unassembled fragments If Save in Subfolder is selected all the results of the assembly will be saved to a new sub folder inside the one containing the fragments This folder will always only contain the assembly results from the one most recent assembly it creates a new folder each time it is run e More Options Under the advanced options you can change the parameters used by Geneious when aligning fragments together These are fully documented if you hover the mouse over them in Geneious To edit these settings you must first chooseCustom Sensitivity in the assembly method panel For sequences which are lower quality or contain many errors or are expected to be divergent from one another you may need to decrease the minimum overlap identity and maximum mismatches per read and increase the maximum gaps allowed per read Choose the options you require and click OK to begin assembling the contig Once complete one or more contigs may be generated If you got more contigs than you expect to get for the se lected sequences then you should try adjusting the options for assembly It is also possible that no contigs will be generated if no two of the sel
237. oo RS OCR SE BARERREEES CORR OE SEE SECE at 201 154 Custom BLAST cres enee e ee ee ee we OO ie Oe Re e 202 16 Workflows 207 16 1 Managing WorklloWS o coea RE RE A e EASES HES 207 16 2 Creating and editing Workflows 50 ES ee REESE SER ES HES 208 CONTENTS 7 16 3 Custom code in Workflows e 212 17 Geneious Education 213 AE I AAA AAA AAA RO AE 213 o EE CIA 214 18 Saving operation settings option profiles 215 19 Collaboration 217 19 1 Mangos Your Accounts ocios pira AAA BS 218 19 2 Manaring Your OA rr a A 220 A AM II 222 19 4 Browsing Searching and Viewing Shared Documents 222 DS Cb A ad hs ees Dh ee A ti de A 223 20 Shared Databases 225 20 1 Supported Database Systems lt lt lt be ke ee es a A ES 225 A POE circos atada ie Shee Rs 226 20 3 Removing a Shared Database coe gc ee ni RA ARA AAA 227 20 4 Administration saa ae a es as ens ea rore re sanaaa da daucan 227 21 Geneious Server 229 21 1 Introduction to Geneious Server 229 21 2 ARTES Genclous Server os o ares AAA A ed 229 21 3 Running jobs and retrieving results aoaaa a 231 21 4 Geneious Server enabled plugins 6 cs eR a a 233 22 Advanced Administration 235 22 1 Default data location 2 0 aos a usan oo eaa a a eae hee i H 235 22 2 Change default preferenc s oe ocs spene rl a pa A AA ESS 235 CONTENTS 22 3 Dpeciiy MOCOS server location oec AR a OE eS 236 244 Deleting builtin pluging oce s AAA AAA AA 237 LE Max MEMON oo
238. or on a few selected sequences choose Design primers on Every Sequence or Selected Sequences in the alignment options at the bottom of the Design New Primers window This option is only available in Geneious 8 1 onwards The best way to save a primer or DNA probe for further testing or use is to select the annota tion for that primer and click the Extract button in the sequence viewer This will generate a separate short sequence document which just contains the primer sequence and the annota tion so it retains all the information on the primer In the case of the reverse primer it will automatically be reverse complemented To delete primers that you don t want just select the primer annotation and click the Delete button You will then be given the option to delete the pair of that primer at the same time 15 1 DESIGN NEW PRIMERS ND2 4 050 CDS 171 4 4 061 F P 050 F Name 4 089 F Type Primer Bind primer_bind Created by primer3 284 4 2 Length 20 4 2 Interval 4 089 gt 4 108 GC 55 0 Tm 60 1 Hairpin Tm 43 6 Self Dimer Tm None Pair Dimer Tm None Sequence CCCAAGCCACAGCATCCATA Product Size 196 Figure 13 3 Primer design output 13 1 10 When no primers can be found If no primers or DNA probes that match the specified criteria can be found in one or more of the sequences then a dialog is shown describing how many had no matches and for what reasons
239. ou are targeting the full sequence or a selection from it Enter the gRNA sequence you want to search for in the Motif panel There is a help box in the Motif section of the options panel that explains how to input your motif and gives an example After selecting your scoring and pairing options press OK to start the operation Geneious will find potential CRISPR targets in your sequence and annotate them onto the document On target Activity Score CRISPR sites can be scored two ways The first is by predicting the on target activity using the method proposed by Doench et al 2014 This scoring algorithm analyses the one and two base features of the gRNA as well as the GC content and uses an experimentally determined predictive model to score the expected activity level of the CRISPR target Scores are between 0 and 1 with a higher score denoting higher expected activity Note that ambiguous bases won t contribute to this score 14 8 CRISPR SITE FINDER 197 Off target interaction analysis The second scoring method compares a CRISPR site with other similar sites and assigns a score based on how individual the CRISPR site is and how likely it is to induce off target inter actions To enable this scoring check Score sites through off target analysis This function uses a scoring system proposed by Zhang et al 2013 which takes into consideration the positions of mismatches between the CRISPR site and its off target sites Scores are between
240. ou chose to return your results in a hit table each search hit is displayed separately in the document table sorted by bit score The bit score gives an indication of how good the alignment is the higher the score the better the alignment In general terms this score is calculated from a formula that takes into account the alignment of similar or identical residues as well as any gaps introduced to align the sequences 204 CHAPTER 15 BLAST Search hits can also be sorted by other columns by clicking on the column header Columns that may be useful to sort by include E value Percent Identity Query Coverage or Grade E value or Expect value represents the number of hits with at least this score that you would expect purely by chance given the size of the database and query sequence The lower the E value the more likely that the hit is real The Grade column is a percentage calculated by Geneious by combining the query coverage e value and identity values for each hit with weights 0 5 0 25 and 0 25 respectively This allows you to sort hits such that the longest highest identity hits are at the top GUNES Query Centric View Virtual Gel Distances Info v Bit Score E Value Grade y Pairwise Id Name Description Hit start Hit end FR M 1 234 79 0 100 0 100 0 X97707 Pongo abelii mitochondrial genome 7 023 7 706 1 202 33 0 99 5 99 0 EU835083 Pongo abelii isolate Orang_45 cytochrom 1 684 1 202 33 0 99 5 99 0 EU835077
241. ow Sequence Selection O Flip View M Highlight Ends Rotate y i i Compute Options o Mm Partition Function _ No GU pairs M Avoid isolated base pairs J Assume circular molecule Dangling Ends Both sides Energy Model RNA Turner 5 Temperature C 3716 Reset Defaults Figure 7 1 A view of an mRNA secondary structure prediction in Geneious 7 2 3D protein structure viewer For molecular structure documents such as PDB documents this displays an interactive three dimensional view of the structure 7 2 1 Structure View Manipulation e Click and drag the mouse to rotate the structure e Hold the Alt or Shift key then click and drag to zoom in out Hold the Ctrl key then right click and drag to pan or if you are using a Mac click and hold press Ctrl and Alt Option then drag to pan 7 2 2 Selection Controls To the right of the structure are controls that let you control the selected part of the structure e If the structure you are viewing contains more than one model the model combo box will you choose between them 7 2 3D PROTEIN STRUCTURE VIEWER Sequence View Text View History Notes MW Reset Color Style B Atoms 4 Bonds 6d Effects Save 3040 v HH gt A 1AA gt 2 EU gt M3 MET E 4 cu A 5 ALA A 6 GLN H 7 His A 8 ALA Mo Leu K 10 Lys IM 11 MET E 12 cu A 13 ALA H 14 HIS Mis LEU Expand All Collapse All To rotate click and drag To zoom hold shift key while
242. own sequence a restriction enzyme or Gateway site or a combina tion of these For more information see section 13 8 A mispriming library is a set of sequences usually repeats which the primers should not bind to Four inbuilt libraries are available for selection or you can upload a custom library of sequences in fasta format For more information on the inbuilt libraries see the Primer3 help page 13 1 8 Batch Primer Design Multiple primer pairs can be designed at once by selecting multiple regions within a single sequence and opening Design New Primers When multiple regions are selected a checkbox Use all selections will appear next to Target and Included regions Check this option for either Target or Included regions to design primers to all selected regions in one step 170 CHAPTER 13 PCR PRIMERS Geneious Primer Characteristics Primer3 Web Interface Primer3 Command Line GC Primer GC PRIMER_ LEFT RIGHT _GC_PERCENT Tm Primer Tm PRIMER_ LEFT RIGHT TM Hairpin Max Self Complementarity Any PRIMER_ LEFT RIGHT INTERNAL_OLIGO SELF_ANY Primer Dimer Max 3 Self Complementarity PRIMER LEFT RIGHTINTERNAL_OLIGO _SELF_END Monovalent Salt Concentration Concentration of monovalent cations PRIMER SALT_CONC Divalent Salt Concentration Concentration of divalent cations PRIMER_DIVALENT_CONC DNTP Concentration Concentration of dNTPs PRIMER DNTP_CONC Sequence Seq PRIMER
243. ping numbering annotation placement and font sizes See section 5 2 8 for more information Yo Statistics Displays statistics about the sequence or alignment currently being viewed such as length molecular weight and nucleotide codon and amino acid frequencies See section 5 2 9 for more information 5 2 6 Sequence Colors The colors of nucleotide and amino acid sequences can be set under the General Options tab by clicking the drop down menu next to Colors Coloring schemes differ depending on the type of sequence For example the Polarity and Hydrophobicity color schemes are available only for protein sequences Alignments and as semblies can be colored by similarity read direction and paired distance if paired reads are used in addition to standard options To change the colors that a particular color scheme uses click the Edit button then click each base and select the new color you wish to use Similarity Color Scheme The similarity scheme is used for quickly identifying regions of high similarity in an alignment In order for a column to be rendered black 100 similar all pairs of sites in the column must have a score according to the specified score matrix equal to or exceeding the specified thresh old So for example if you have a column consisting of only K Lysine and R Arginine and are using the Blosum62 score matrix with a threshold of 1 then this column will be colored entirely black because the Blosu
244. pper followed by an op tional fine tuning step to better align reads around indels to each other rather than the reference sequence Various optimizations and heuristics are applied at each stage but a general outline of the algorithm is 140 10 CHAPTER 10 ASSEMBLY AND MAPPING First the reference sequence s is indexed to create a table making a record of all locations in the reference sequence that every possible word series of bases of a specified length occurs Each read is processed one at a time Each word within that read is located in the reference sequence and that is used as a seed point where the matching range is later expanded outwards to the end of the read Ifa read does not find a perfectly matching seed the assembler can optionally look for all seeds that differ by a single nucleotide Before the seed expansion step all seeds for a single read that lie on the same diagonal are filtered down to a single seed During seed expansion when mismatches occur a look ahead is used decide whether to accept it as a mismatch or to introduce a gap in either the reference sequence or read The mapper handles circular reference sequences by indexing reference sequence words spanning the origin and allowing the expansion step to wrap past the ends P 8 8 8 pP P PP All results are given a score based on the number of mismatches and gaps introduced Normally the best scoring or a random one of equal
245. pports the import of several Vector NTI formats e gb and gp formats These formats are used in Vector NTI for saving single nucleotide and protein sequence documents They are very similar to the GenBank formats with the same extensions although they contain some extra information e apr format This format is used for storing alignments and trees made with AlignX Vector NTT s alignment module e maf pa4 0a4 ea4 and ca6 formats These are the archive formats which Vector NTI uses to export whole databases e cep format This format is produced by the ContigExpress module and Geneious will import sequences including the positions of the base calls traces qualities trimmed regions annotations and editing history for individual reads and contigs 3 3 IMPORTING FILES FROM PUBLIC DATABASES 41 3 3 Importing files from public databases Geneious is able to communicate with a number of public databases hosted by the National Centre for Biotechnology Information NCBI as well as the UniProt database You can access these databases through the web at http www ncbi nlm nih gov and http www uniprot org respectively These are all well known and widely used storehouses of molec ular biology data more information on each is given in the sections below You can search these databases through Geneious by selecting the NCBI or Uniprot database folders at the bottom of the Sources tree and entering your search term Pre
246. provides several operations for designing and working with PCR Primers and DNA or hybridisation probes PCR Primers and DNA or hybridisation probes can be designed for or tested on existing nucleotide sequences or alignments A PCR product can be extracted from a sequence that has been annotated with both a forward and a reverse primer 5 extensions con sisting of restriction enzymes or arbitrary sequence may also be added to primer documents In addition Geneious can determine the primer characteristics for a primer sized sequence and convert it into a primer Characteristics can also be determined for any number of primer sized selections made in the Sequence View To use any one of these primer operations simply select the appropriate nucleotide sequences and either select Primers from the Tools menu or right click Ctrl click on Mac OS X on the document s and select Primers A popup menu will appear showing the operations valid for your current selection 13 1 Design New Primers Geneious uses Primer3 to design PCR primers The Primer Design dialog allows you to set options for where your PCR primers should sit what size product to return and characteristics such as primer length and melting temperature Two options are available for primer design Design New or Design with Existing Design New designs a pair of forward and reverse primers You can specify if you wish to design with or without a matching probe Design with Existing can desi
247. quence View Quality The quality scores associated with a chromatogram can be viewed by checking the Qual box under the Chromatogram graph options This displays a quality measure typically Phred quality scores for each base as assessed by the base calling program The quality is shown as a shaded blue bar graph overlaid on top of the chromatogram Note that those scores represent an estimate of error probability and are on a logarithmic scale the highest bar represents a one in a million 1076 probability of calling error while the middle represents a probability of only a one in a thousand 1073 Sequence Logo When checked bases letters are drawn in size proportional to call quality where larger implies better quality or smaller chance of error Note that the scale is logarithmic the largest base represents a one in a million 107 or smaller probability of calling error while half of that represents a probability of only a one in a thousand 1073 On large contigs over 100 000 bp long the sequence logo can t be efficiently calculated in regions of over 1000 fold coverage in which case the sequence logo will display To view the raw chromatogram traces click the Chromatograms tab above the sequence viewer In this view the exact location of the base call can be viewed by checking Mark calls To view sequence logos indicating base quality in this view check Scale by confidence The Trace op tions for X and Y scales allow you to
248. quences in the alignment with the exception of the reference sequence 9 3 1 Highlighting Identical or variable sites in an alignment can be highlighted by checking the Highlighting option under the Display tab The drop down menus allow you to choose what to highlight and whether the consensus or reference sequence should be used for the comparison The options for what to highlight are e Disagreements This greys out residues that are identical to the consensus or reference in that column allowing you to quickly locate variable sites in the alignment e Agreements Greys out residues that are not indentical to the consensus or reference allowing you to quickly locate conserved sites in the alignments e Ambiguities Greys out non ambiguous residues 120 CHAPTER 9 SEQUENCE ALIGNMENTS e Gaps Greys out non gap positions e Transitions transversions Greys out residues that are not transitions transversions com pared to the consensus or reference sequence When highlighting transitions transversions it is recommended you turn on the ignore gaps consensus option or some residues may be wrongly highlighted due the consensus displaying N for sites that contain gaps and non gaps Go to next disagreement agreement transition transversion ambiguity goes to the next high lighted feature as described in the previous section If Use dots is checked non highlighted residues are displayed as dots instead of being greyed out 9 3
249. r and will be ignored when primer testing is conducted against potential target sequences If the primer is annotated onto a sequence following testing the extension sequence is shown in the list of the annotation s qualifiers If the primer or a PCR product is extracted from this annotation the result will include the extension 13 9 Extract PCR Product Once primers are annotated on a sequence the resulting PCR product can be extracted by selecting Primers Extract PCR Product If only a single pair of primers are annotated on a sequence then these will automatically be chosen as the Forward and Reverse primer If multiple primers are annotated on a sequence then the drop down menus allow you to choose which primers to use for extracting a single PCR product or alternatively you can choose to Extract PCR products from all primers 178 CHAPTER 13 PCR PRIMERS 5 500 5 510 5 520 5 530 5 540 5 553 5 560 5 570 ATGTTATTGTCACAG CGCCTTTGTAATAATCTT CTT TATAGTTATG CCAATTAT TTGGAGGCTTTGGAAACTGN MammothCOX1_F _ cox1 cos x Name MammothCOX1_F Type Primer Bind primer_bind Created by primer3 Length 20 Interval 5 498 gt 5 517 Product Size 250 Mismatches 0 eens YGC SO 0 Tm 60 0 Hairpin Tm 39 8 Self Dimer Tm None Pair Dimer Tm None Sequence TATTGTCACAGCACACGCCT Figure 13 8 Primer annotation with 5 extension 15 10 MORE INFORMATION 179 When you click OK a new sequence document is produced cont
250. r of users to run jobs on Geneious Server simul taneously If your user account has its own access license GSAL then you can connect to the server and execute jobs immediately without having to wait for a queue license to become available If your account doesn t have an access license then you can log in and submit the job to the server where it will join the queue and execute when a queue license becomes available 21 2 Accessing Geneious Server Assuming you have your account configured on the server you 1l need to install the necessary Geneious Server plugins Many of your normal Geneious plugins are already server aware but there are other plugins which are different from the standard plugins or are exclusive to Geneious Server Your administrator can provide you with a download location for Geneious Server plugins You can get them either from the Geneious Server itself or they may be hosted on a network 233 234 CHAPTER 21 GENEIOUS SERVER location with the gplugin files If your institute has a web interface get the URL from your administrator and you should see a page like figure 21 1 geneious SERVER Download the client plugin file Install this file into your copy of Geneious to access Geneious Server Download the BWA plugin file Install this file into your copy of Geneious to install BWA plugin Download the Bowtie plugin file Install this file into your copy of Geneious to install Bowtie plugin Download the
251. r protocol click on that contact and select New Chat Session either from the Collaboration submenu or from the popup menu right click on the contact or Ctrl click on Mac OS X Type your messages into the text field at the bottom of the window that pops up and click Send or press the Enter key to send 19 5 2 Chatting with Multiple Contacts Starting a Chat Session with Multiple Contacts To invite several contacts to join you in a new chat session click on your account not the contacts and then select New Chat Session from either the Collaboration submenu or the context menu right click on the account or Ctrl click on Mac OS X Select the online con tacts which you want to invite you can select a range by Shift clicking or add contacts to the selection by Ctrl clicking Click invite to create this new chat session Accepting or Declining an Invitation to Chat When one of your contacts invites you to chat a dialog will appear asking you to accept or decline the chat invitation Clicking Accept will open a chat window that will allow you to chat with the contact who invited you and with all other contacts that were invited If you decline that invitation and enter a reason optional this reason will be displayed to everyone in the chat Sending and Viewing Messages in the Chat The chat window displays your own and your contacts previous messages You can enter new messages in the field at the bottom These m
252. rated Values osv tsv Spreadsheet files Microsoft Excel SAM BAM sam bam Contigs SAMtools Sequence Chromatograms abl scf Raw sequencing trace amp sequence Sequencing machines VCF VCE Annotations 1000 Genomes Project Vector NTI sequence gb gp Nucleotide amp protein sequences Vector NTI Vector NTI AlignX alignment apr Alignments Vector NTI AlignX Vector NTI Archive ma4 pa4 oa4 Nucleotide amp protein sequences ead ca6 enzyme sets and publications Vector NTI Vector NTI ContigExpress cep Nucleotide sequence assemblies Vector NTI Vector NTI database VNTI Database Nucleotide amp protein sequences enzyme sets and publications Vector NTI BED annotations The BED format contains sequence annotation information You can use a BED file to anno tate existing sequences in your local database import entirely new sequences or import the annotations onto blank sequences CLUSTAL alignment The Clustal format is used by ClustalW and ClustalX two well known multiple sequence align ment programs 3 2 DATA INPUT FORMATS 35 Clustal format files are used to store multiple sequence alignments and contain the word clustal at the beginning An example Clustal file CLUSTAL W 1 74 multiple sequence alignment seql KSKERYKDENGGNYFOLREDWWDANRETVWKAITCNA
253. re if their mates also score well against each other Similarly a sequence and its mate will be given a higher score if they both align at approximately their expected distance apart to an already formed contig The effect of this heuristic is that paired read de novo assembly starts out by finding 2 sets of paired reads and forming 2 contigs Each of these 2 contigs will contain 1 sequence from each pair and the 2 contigs are expected to be separated by the expected mate distance Assembly proceeds from there either adding new paired reads to the contigs or forming new pairs of contigs which eventually merge together Due to the nature of this algorithm paired read de novo assembly in Geneious only works well if you have high coverage of paired reads a hybrid assembly of mostly unpaired data with a few paired reads will not make good use of the paired read data but this is expected to improve in future versions 4 Each contig generated by a gapped de novo assembly has some minor fine tuning per formed on it both during assembly and upon completion For each gapped position in a 136 CHAPTER 10 ASSEMBLY AND MAPPING sequence a base adjacent to the gap is shuffled along into the gap if it is the same base as the most common base in other sequences in the contig at that position After doing this if any column now consists entirely of gaps that column is removed from the contig 5 Other minor heuristics are applied throughout the assembly to impro
254. re kog ak ee a ES EA AAA RA A AA 237 22 6 Web Linking to Data in Geneious 0 2000 a ad 237 Chapter 1 Getting Started The best way to get started with Geneious is to try out some of our tutorials The Tutorial option under the Help menu in Geneious provides an inbuilt tutorial with a basic introduction to the major features of Geneious Additional tutorials on specialized functions can be downloaded from our website http geneious com tutorials For additional information and help with troubleshooting please visit the Geneious support website at http support geneious com 1 11 Downloading amp Installing Geneious Geneious is free to download from http geneious com download If you are using Geneious for the first time you will be offered a free trial If you have already purchased a license you can enter it when Geneious starts up To download the latest version of Geneious click on http geneious com download or type it in to your internet browser choose the version of Geneious you want to download and click Download If you have a 64 bit Linux or Windows machine ensure you check the 64 bit box Geneious is compatible with the three most common operating systems Windows Mac and Linux Check that you have one of the following OS versions before you launch Geneious Operating System System requirements Windows XP Vista 7 8 10 Mac OS 10 6 10 7 10 8 10 9 10 10 Linux CentOS 6 RHEL6 Ubuntu Desktop LTS
255. really a variant here In other words the probability of seeing 2 G s by chance due to a sequencing error plus the probability of seeing 3 G s by chance due to a sequencing error which is calculated using the binomial distribution 3 C 0 01 0 99 C3 0 013 Ck is a binomial coefficient False SNPs due to strand bias when sequencing errors tend to occur only on reads in a single direction can be eliminated by specifying a value for the Minimum Strand Bias P value set ting A Strand Bias P Value property is added to each SNP to indicate the probability of seeing a strand bias at least this extreme assuming that there is no strand bias SNPs with a smaller strand bias p value will be excluded from the results when using this setting 11 1 FINDING POLYMORPHISMS 147 Click the up arrow next to the exponent of the Minimum Strand Bias P Value setting to in crease the number of variants found If there are any forward reverse or reverse forward style paired reads then variants with strand bias which are less than 1 5 times the insert size from either end of the contig will not be filtered out Results display The results of the Variant SNP finder are added to the reference sequence in the assembly or alignment as an annotation track Clicking Save and clicking Yes when prompted to apply the changes to the original sequences will add this annotation track onto the original reference sequence file If there is no reference sequen
256. reating viewing and editing sequences 57 Dl Creating few seguents costaria AA A PES 57 S2 ICON eet Shee Oo oe ERR ED OS PEERS ee Be OS 58 Die TIRE SEUA 2 es ER BER ERE Ed BA Lew ES 71 5 4 Complement and Reverse Complement 2 04 2528 2 nes 73 Be Ter Se III 73 S6 VICI CRORES cio iee ppe Ee we AE ARA a eS 75 S7 Metadata eccu eb pions atei an p IAE 77 Parent Descendant tracking 81 61 Editing Linked Documents oe caocca aa EE a 83 62 The Lineage en 5 he Kae Sree BS OS RS OEE O ATA A 84 RNA DNA and Protein structure viewer 87 7 1 RNA DNA secondary structure fold viewer 2 0 0 0 0 ooo 87 7 2 oD protein structure WIOWED ec oe e se Oe EOS Be Bede Bde Es 88 Working with Annotations 91 8 1 Viewing editing and extracting annotations o ooo o 91 82 Adding AO rar de A Eee AE AAA 97 CONTENTS 9 10 11 12 13 8 3 Compare Annotations Sequence alignments 91 Dotplols lt lt 220 dane es 9 2 Sequence Alignments 9 3 Alignment viewing and editing 9 4 Consensus sequences Assembly and Mapping 10 1 Read processing s8i 404 10 2 De novo assembly 10 3 Map to TAE oe ek ee 10 4 Viewing Contigs 10 5 Editing Contigs iia 10 6 Extracting the Consensus Analysis of assemblies and alignments 11 1 Finding polymorphisms 11 2 Analyzing Expression Levels Building Phylogenetic trees 12 1 Phylogenetic tree representation 12 2 Tr
257. rect amp Normalize Reads from the Sequence menu This function uses BBNorm from the BBtools suite and it requires Java 7 or later in order to run For a detailed explanation of any BBNorm setting hover the mouse over the setting or click the help question mark button next to the custom options under More Options For more information see the BBNorm page on SeqAnswers 10 1 4 Setting paired reads To assemble paired read or mate pair data prior to assembly you first need to tell Geneious the reads are paired The assembler will then automatically used the paired data unless you turn off the advanced option to Use paired distances To set up paired reads you need to select the document s containing the paired reads and se lect Set Paired Reads from the Sequence menu Depending on your data source reads could be in parallel sets of sequences or interlaced so you need to tell Geneious which format Geneious will guess and select the appropriate option based on the data you have selected so most of the time you can just use the default value for this However you must make sure you select the correct Relative Orientation for your data Different sequencing technologies orientate their paired reads differently All paired read data will have a known expected distance between 130 CHAPTER 10 ASSEMBLY AND MAPPING each pair It is important you set this to the correct value to achieve good results when assem bling If you don t kno
258. rent document in a separate window This allows you to have several documents open at once and gives more space for viewing This can also be achieved by double clicking in the document table P Help Opens the Help Panel section 2 4 and displays some short help for the current viewer 2 4 The Help Panel The Help Panel has a Help tab and a Tutorial tab The Help tab provides you with information about the service you are currently using or the viewer you are currently viewing The help displayed in the help tab changes as you click on different services and choose different viewers 26 CHAPTER 2 THE GENEIOUS MAIN WINDOW The Tutorial is aimed at first time users of Geneious and has been included to provide a feel for how Geneious works It is highly recommended that you work through the tutorial if you haven t used Geneious before The Help panel can be closed at any time by clicking the button in its top corner or by toggling the Help button in the Toolbar O Help Return to contents Sequence alignment If the sequences you just imported are not selected select them all now To align all the sequences click on the Align Assemble button in the toolbar and choose Mu tiple Align Click Oto accept the default alignment settings When the alignment has finished a new alignment document will be added to the current folder and selected The alignment will be displayed in the sequence viewer If the ali
259. residues are variable A consensus is constructed from the most frequent residues at each site alignment column so that the total fraction of rows represented by the selected residues in that column reaches at least a specified threshold IUPAC ambiguity codes such as R for an A or G nucleotide are counted as frac tional support for each nucleotide in the ambiguity set A and G in this case thus two rows with R are counted the same as one row with A and one row with G When more than one nu cleotide is necessary to reach the desired threshold this is represented by the best fit ambiguity symbol in the consensus for protein sequences this will always be an X For example assume a column contains 6 A s 3 G s and 1 T If the consensus threshold is set to 60 or below then the consensus will be A If the consensus threshold is set to between 60 and 90 then the consensus will be R If the consensus threshold is set to over 90 then the consensus will be D 9 4 CONSENSUS SEQUENCES 123 In the case of ties either all or none of the involved residues will be selected For example if the above case instead had 6 A s 2 G s and 2 T s then for a consensus threshold of 60 or below an A will be called Above a threshold of 60 a D will be called When Ignore Gaps is checked the consensus is calculated as if each alignment column consisted only of the non gap characters otherwise the gap character is treated like a normal residue
260. roperties and intervals can be edited see Figure 8 3 To move the annotation onto a track click the Track option and either choose an existing track or type in the name of a new track you want to create To move the annotation from a track to the sequence itself choose No track in this setting In the Properties section properties can be added edited removed or moved up and down in the list by clicking the buttons to the right The annotation color can also be set in this section by clicking the color boxes The Override color sets the color for that particular annotation only but does not change the color of other annotations of that type To change the color of all annotations of that type click the Color box The Intervals section shows where the annotation is located on the sequence To change the location the direction or mark an annotation as partial select the interval and click Edit The direction of the annotation can be changed by clicking the colored arrow Check truncated left 8 1 VIEWING EDITING AND EXTRACTING ANNOTATIONS 99 e0 Edit annotation in ygh CDS divergent reference Type CDS A Track No Track gt Direction e Forward _ Reverse _ Undirected y Properties Type Color Override Color gt lt gene ygh Mo Move Up locus_tag ECIAI1_3114 function 14 Cell envelope inference ab initio prediction AMiGene 2 0 note Evidence 2b Function of strongly homologous gene PubMedid 164
261. roups of the se quence For a 5 triphosphate group weights are adjusted using Molecular Weight calculated molecular weight 159 0 e Isoelectric Point Calculates the isoelectric point of a protein as per this method but using the following values for the amino acids D 3 9 E 4 1 C 8 5 Y 10 1 H 6 5 K 10 8 R 12 5 74 CHAPTER 5 CREATING VIEWING AND EDITING SEQUENCES Extinction Coefficient Calculates the extinction coefficient of a protein as per this paper using the following values for the amino acids and assuming all cysteines are paired in a disulfide bridge making cystine C 62 5 only counting up to an even number W 5500 Y 1490 Statistics for multiple sequences lists alignments assemblies Sequences The number of sequences in the document or in the currently selected region Identical sites When viewing alignments or assemblies this considers only those columns in the alignment that have at least 2 nucleotides amino acids gaps that are not free end gaps and are not columns consisting entirely of gaps A column not meeting this re quirement is not even counted as non identical for the percentage calculation A column meeting this requirement is considered identical if it contains no internal gaps and all the nucleotides amino acids are identical Ambiguity characters are not interpreted so a nucleotide column of A and R is not considered identical Pairwise Identity When viewing alignments or assemblies thi
262. rting profiles See sharing profiles section below for more on import and export Sharing profiles There are two ways to share option profiles Import and export from the Manage Profiles window allows you to save a file containing a particular profile These can be emailed to other Geneious users and imported for use with their data The easiest way to import a profile is by dragging the file directly in to Geneious If a profile is marked as Shared when it was created or by editing it then the profile will be copied across to any Shared Database that you connect to This means anyone else who connects to the same Shared Database will automatically have the profile under their Load Profile menu Note Once a profile is shared it cannot be un shared but it can be deleted Also other users can edit or delete a shared profile at any time Chapter 19 Collaboration Collaboration is an external plugin Collaboration allows Geneious users to share the products of their research and work with each other Based on an open Internet protocol called XMPP or Jabber it allows you to maintain a list of contacts so that you see who is online when you sign on yourself You can then share documents with your online contacts and browse and work with their documents in return The list of contacts is stored on the server so you can easily access an account including its contacts both at work and on your private computer Collaboration can work wit
263. ry of an document can be viewed by going to the Info tab above the sequence viewer and choosing History This displays information on how the document was created plus a record of each time it has been modified The exact information displayed is flexible but is the entries will always include the time and user responsible for the edit An entry may also reference other documents via hyperlinks and has the ability to display a re creation of the options used Saving of history can be disabled for performance or privacy reasons by going to the Appear ance and Behaviour tab in Preferences see section 1 2 1 Chapter 5 Creating viewing and editing sequences 5 1 Creating new sequences New sequences can be imported from existing files as described in chapter 3 or they can be created manually by going to Sequence New Sequence Here you can paste or type in the residues for your new sequence then enter the Name Description and Organism for your sequence if required see figure 5 1 Geneious will automatically determine whether your sequence is nucleotide or protein based on the composition of the bases you enter You can change this by clicking the Type option If your sequences are oligonucleotides choose Primer or Probe as the type To create a new sequence from an existing sequence select the region of sequence that you want then click the Extract button above the sequence viewer or go to Sequence Extract Regions This will cr
264. s residues in each position For example if one sequence is LLK and the other is LIK using the Blosum62 matrix L L scores 4 I I scores 4 L I scores 2 K K scores 5 Therefore the total score is 4 2 5 11 out of a maximum of 4 maximum 4 4 5 13 for a percentage similarity of 85 Gaps if allowed are scored as the lowest value from the score matrix e g 4 for Blosum62 Annotate from Database Annotate from Database allows you to annotate your sequence with particular genes or mo tifs from a custom annotation database The annotation database is a folder called the Source folder within your Geneious database which contains sequences with existing annotations e g specific genes you are interested in searching for on other sequences This function uses a BLAST like algorithm to search for annotations in the Source folder that match your sequence Annotations which match with the given similarity are copied to your sequence The default annotation folder is the PlasMapper Features folder from the Sample Documents which con tains features from the database used by PlasMapper By default this also annotates Gateway recombination sites This can be turned off in the Advanced options To use this function 1 Create your annotation database by placing your sequences containing annotations of interest in a separate folder in Geneious 2 Select the sequence s you want to annotate and check Annotate From in the Live Annotate and Predict tab in
265. s to define the insert boundaries for the Golden gate recombination reaction These are used in the order of precedence listed below Rules 1 6 and if required Geneious will design a primer for PCR amplification of the insert to generate the required cut site Rules in order of precedence 1 Existing type IIS cut site s If Geneious detects a pair of appropriately orientated type IIS sites with unique overhangs then it will assume you wish to use them Geneious will also assume that you have DNA available to use and so will not design primers for PCR If only one type IIS cut site is detected then Geneious will design a primer that incorporates the site and a second opposite orientation primer for PCR will be designed based on rules 3 6 2 Existing Overhang s If Geneious detects a pair of valid overhangs compatible with the specified type IIS site then it will assume you wish to use them Geneious will also assume that you have this sticky ended DNA available and so will not design primers for PCR 3 Existing primer_bind annotation s with valid type IIS cut site s on the extension If Geneious detects a pair of inward facing primer_bind annotations with valid compati ble type IIS sites then it will assume you wish to use them Geneious will assume that you already have the corresponding primers and new primers will not be designed for the region If Geneious detects a single primer_bind annotation with a suitable type II
266. s gives the average per cent identity over the alignment This is computed by looking at all pairs of bases at the same column and scoring a hit one when they are identical divided by the total number of pairs Ambiguity characters are interpreted meaning a nucleotide A vs a nucleotide R is considered to have 50 identity For both Identical sites and Pairwise Identity the statistics are calculated from the subset of sequences and nucleotides amino acids selected If just a single sequence is selected the statistics are calculated as if all sequences are selected over the selected columns The consensus sequence is always excluded from calculation of both of these values Coverage of Bases When viewing a contig assembly this gives the mean standard devi ation minimum and maximum of the coverage of each base in the consensus sequence For small contigs the coverage is further broken down into coverage by reads mapped to the forward and reverse strands For large contigs separate forward reverse coverage can t be efficiently calculated so is displayed as If your contig has a reference sequence then the percentage of the ungapped reference sequence that is covered by at least 1 read is also displayed Selecting a sub region of your contig will display statistics for just that region including calculation of separate forward reverse coverage on large contigs Ungapped Lengths of Sequences Displays the mean standard deviation min
267. s online import citations perform searches on your own notes and insert references into documents It also generates a bibliography in different styles Geneious can interoperate with EndNote using Endnote s XML Extensible Markup Language file format to export and import its files FASTA sequences The FASTA file format is commonly used by many programs and tools including BLAST T Coffee and ClustalX Each sequence in a FASTA file has a header line beginning with a gt followed by a number of lines containing the raw protein or DNA sequence data The sequence data may span multiple lines and these sequence may contain gap characters An empty line may or may not separate consecutive sequences Here is an example of three sequences in FASTA format DNA Protein Aligned DNA gt Orangutan ATGGCTTGTGGTCTGGTCGCCAGCAACCTGAAT CTCAAACCTGGAGAGTGCCTTCGAGTG gt gi 532319 pir TVFV2E TVEV2E envelope protein ELRLRYCAPAGFALLKCNDADYDGFKTNCSNVSVVHCTNLMNTTVTTGLLLNGSYSENRT QIWOK gt Chicken CTACCCCCCTAAAACACTTTGAAGCCTGATCCTCACTA CTGT CATCTTAA FASTQ sequences FASTQ format stores sequences and Phred qualities in a single file GenBank sequences Records retrieved from the NCBI website http www ncbi nlm nih gov can be saved in a number of formats Records saved in GenBank or INSDSeq XML formats can be imported into Geneious 3 2 DATA INPUT FORMATS 37 Geneious format The Geneious format can be used to store all
268. search inter val Delete Delete the agent permanently Any documents retrieved by the agent will remain in your local documents 48 CHAPTER 3 IMPORTING AND EXPORTING DATA 3 5 Exporting files To export files from Geneious select the file or files you want to export and click File Export Selected Documents Each data type has several export options as detailed in the table below Any set of documents may be exported in Geneious native format and these files are back compatible to Geneious version 6 0 e g Files exported in geneious format from Geneious R7 or R8 can be imported to Geneious R6 or later If you wish to export the parents and or descendants of a particular file at the same time choose File Export with Parents Descendants Data type Export format options DNA sequence FASTA Genbank XML Genbank flat Geneious Amino acid sequence FASTA Genbank XML Genbank flat Geneious Chromatogram sequence ABI Geneious Sequence with quality FastQ Qual Geneious Annotation GFF BED Geneious Multiple sequence alignment Phylip FASTA NEXUS MEGA Geneious Assembly Phrap ACE Geneious SAM BAM Variant calls VCF single sample only Phylogenetic tree Phylip FASTA NEXUS Newick MEGA Geneious PDF document PDF Geneious Publication EndNote 8 0 Geneious Graphs CSV WIG Document Properties CSV TSV Geneious Documents imported in any chromatogram or molecular structure format can be re exported
269. second option allows you to specify multiple primers and probes to test all selected se quences against Click the Choose button then hold down CTRL click command click on Mac to select multiple primers and probes from many different locations in your database Alterna tively you can select one or more folders to test with all the primers and probes inside them or click the Use All button to use every primer in your database As with the first option you can choose which types of primers you d like to test for by select ing the checkboxes on the left Note that each primer you select will be considered in both the forward and reverse roles if you have checked both Search for Forward and Search for Reverse One final checkbox Pairs Only forces primers and probes to be considered as pairs with the probe inside otherwise they can be found anywhere in the sequence with no constraints 176 CHAPTER 13 PCR PRIMERS All of the same options available for designing primers also apply to testing so if the primers are expected to bind to quite different regions of the test sequences the primer binding region may have to be extended and the target region option can be omitted By default only primers that match the target sequence exactly will be found If you wish to allow a limited number of mismatches between the primer and target sequence you can specify this under Maximum Mismatches You can limit the position in which mismatches are allowed b
270. select Nucleotide similarity search or Protein similarity search and enter the sequence text Geneious will try to guess the type of search based on the text so that simply entering or pasting a sequence fragment may change the search type automatically The search locates documents containing a similar string of residues and orders them in de creasing order of similarity to the string The ordering is based on calculating an E value for each match You can read more about the E value in chapter 15 For the search to be successful you need to specify a minimum of 11 nucleotides and 3 amino acids Note that search times depend on the number and size of your sequence documents and so may take a long time to complete 4 2 3 Find in Document The Find in Document option under the Edit menu allows you to search for a particular motif in your sequences annotations or document names For example you can search for a partic ular string of nucleotides in sequences or alignments or search for annotations and sequences by name or number The search can optionally be made case sensitive Matching regions are selected in the document at the end of the search The shortcut for this function is Ctrl F To find the next match for the text specified in the dialog you can use F3 or Ctrl G and to find the previous match use Ctrl Shift G or Shift F3 4 3 FIND DUPLICATES 57 4 2 4 Filtering and Similarity sorting The Filter S allows you to instantly
271. ser table beforehand You will want to give yourself the role of Admin for the Everybody group if you want to perform administrative functions within Geneious Unfortunately at this time there is no interface for assigning groups and roles to users So you will need some knowledge of SQL in order to take advantage of this feature You can create groups by adding entries into the g group table in the database Assign users groups and roles in the table g_user_group_role It is likely that if you are running in a multi user environment and taking advantage of groups and roles you will want to give only read access of the table g user_group role to your users This is so your users can not edit this table with SOL directly as you would do You will also want to add all of your users into g user manually so Geneious does not think that they are first time users and fail trying to insert them into the Everybody group due to read only access 232 CHAPTER 20 SHARED DATABASES 20 4 2 Document Compatibility Between Versions By default the Shared Database stores documents in the latest document format the same way the Local Database does This can cause compatibility problems if different sets of users are using different versions of Geneious For example if a user from Geneious 8 1 saves a new alignment document users from Geneious 6 will be unable to read those documents To get around this issue the Shared Database can be configured to save docume
272. sitivity will be auto Minimum BLAST alignment score B All Oligos in Database C Trim primers M Allow Mismatches Minimum Match Length M Error Probability Limit 8 decrease to trim more Trim regions with more than a 5 chance of an error per base C Maximum low quality bases ofle _ Maximum ambiguities 2 8 M Trim 5 End C At least 0 5 bp Y Trim 3 End C At least 0 2 bp C Maximum length after trim 1 000 l Trim excess from 3 end a Cora Oe Figure 10 2 Trimming options 128 CHAPTER 10 ASSEMBLY AND MAPPING e Remove new trimmed regions from sequences Calculate new trimmed regions and remove them from the sequence s completely This can be undone in the Sequence View before the sequences are saved Remove existing trimmed regions from sequences This is only available when there are already trimmed regions on some of the sequences This will remove the existing trimmed regions from the sequences permanently no new trimmed regions are calcu lated Trim vectors Screens the sequences against UniVec or your own custom BLAST database to locate any vector contamination and trim it This uses an implementation similar to NCBI s VecScreen to detect contamination http www ncbi nlm nih gov projects VecScreen Multiple databases can be selected to trim from by clicking the sign e Trim primers Screens the sequences against primers in your local database Error Probability Limit
273. ss Enter or the Search button to initiate the search If you get a connection error you may need to configure your network connections manually as described in Section 1 2 5 For advanced search options click the More Options button This allows you to search for spe cific terms in specific fields of the Genbank or Uniprot documents such as specific organisms or author names By clicking the icon you can search in multiple fields at once and choose to match either Any of the fields if only one of the fields needs to match or All of the fields if all of the fields must match For more on advanced search options see section 4 2 If you have a list of known accession numbers that you wish to download you can enter these in the Search box separated by a comma For consecutive accessions enter the first and last numbers separated by a colon and append acen to this E g Entering AB000001 AB000009 accn will download all accessions between AB000001 and AB000009 The results will appear in the Document table as they are found The Search button changes to a Stop button while the search is running and this can be clicked at any time to terminate the search As the results are downloaded you will see a small padlock icon in the status bar above the Document Table which indicates that these items cannot be modified in any way You must drag the file into a folder in your local database if you wish to retain the file a
274. sting data across to a new location or open an empty database at that location Geneious will remember this new location when you exit eD0 Preferences Plugins and Features Appearance and Behavior Keyboard NCBI Sequencing Data Storage Location Users hilary Geneious 8 0 Data Browse Search History Clear Y Check for new versions of Geneious Also check for beta versions of Geneious Check for updates now Figure 1 1 Setting the location of your local documents Note that if you uninstall Geneious your data will not be wiped as the application itself is stored in a different location from the data folder 1 2 3 Sharing files or the local database The best solution for sharing files with other users is to set up a shared database see Chapter 20 in a central location which all users can connect to Alternatively you can share files using the collaboration plugin see Chapter 19 but users all need to be running Geneious at the same time for the files to be accessed You can also export documents in geneious format and transfer these files via a USB or network drive for others to use 14 CHAPTER 1 GETTING STARTED We do not recommend storing databases on Dropbox or network drives as a way of sharing files 1 2 4 Changing the memory available to Geneious Geneious runs in a Java Virtual Machine When this JVM starts it will be allocated a certain amount of RAM and the program can use less than that but never mor
275. synonymous set Use threshold of 0 to only replace codons that code for the wrong amino acid in the target genetic code or that have to be changed to avoid restriction sites Avoid restriction sites lets you select a set of enzymes that you intend to use to cut the target sequence If you select an enzyme set with this option the result sequence will not include any sites that match a recognition sequence in the set The enzyme set choices are the same as are used in 14 1 After configuring your options click OK to start the analysis and annotate the optimized codons on the sequence or create the new sequence document 200 CHAPTER 14 CLONING Annotate Codons Optimize and Save As Optimized Human proinsulin Source Genetic Code Target Organism Escherichia coli k12 igh Target Genetic Code Bacterial o Threshold to be rare 8 C Avoid restriction sites commonly used enzymes E Figure 14 7 Codon Optimization options dialog Chapter 15 BLAST BLAST stands for Basic Local Alignment Search Tool Altschul et al 1990 It allows you to query a sequence database with a sequence in order to find entries in the database that contain similar sequences When BLAST ing you are able to specify either nucleotide or protein sequences and nucleotide sequences can be either DNA or RNA sequences Sequences can be BLAST ed against databases held at NCBI see NCBI BLAST or contained within your local Geneious data
276. t CpG per match GTYARAC blunt CpG EcoBl 1 per match AMAGCTT 5 4 nucleot EcoBl per match GAANTC 5 3 nucleot per match GTTAAAC blunt CpG EcoKI per match CACGG 5 2 nucleot CpG per match GGTACAC 3 4 nucleot per match Mba AGAIC gt un 6 6 6 nda E HE A HE gt gt ba un oooocco ts S 4 nicleot Dam CnC E ner match Restriction enzyme information was obtained from rebase Restore Defaults Figure 14 1 Find Restriction Sites restriction enzymes table accessible under the Advanced option 14 2 DIGEST INTO FRAGMENTS Cut Positions _ Annotated restriction sites Selected Enzyme set position 124 commonly used enzymes Cut Using e Each enzyme separately Q All enzymes at once M Extract to sequence list document 52 enzymes selected Y Minimum recognition sequence length Co 4 8 C Only include enzymes matching 1 8 to 2 8 times Cut Anywhere a C Only consider enzymes with GACGTAC GTAMKAC GATGCAC CAYCGRG GAGWCC TGGACCA GAGATCC GCCNNNNAN AAGATCT GAGTNACC O EE Figure 14 2 Digest into fragments options dialog with extended options showing _124 and 123 2 palindromic 3 4 nucleoti 5 2 nucleoti blunt 5 4 nucleoti 5 4 nucleoti 5 3 nucleoti blunt 5 4 nucleoti 3 nucleoti 4 nucleoti 5 nucleoti S 1 nucleoti recognition sequence CpG CpG
277. t and Reverse Complement To display the complement of a sequence alongside the original sequence check the Comple ment box in the Display tab To display only the complement and not the original nu cleotide sequence uncheck Nucleotides Note that these options are only for display purposes if you wish to create a separate document to work with the complement not reverse comple ment of a sequence you will need to install the Complement or Reverse plugin by going to Tools Plugins Once the plugin is installed go to Sequence Complement only to create a new document containing the complement To reverse complement a nucleotide sequence i e reverse the sequence direction and replace each base by its complement click the R C button above the sequence viewer or go to Reverse Complement under the Sequence menu You can also access this option right clicking in the sequence viewer and selecting it from the popup menu When you click Save after reverse complementing the tag reversed will be added to the sequence name When only part of a sequence is selected you can choose to either reverse complement only the selected region and extract it to a new sequence document or reverse complement the entire sequence On alignment or contig documents you can reverse complement individual sequences within the alignment or assembly by selecting that sequence and choosing reverse complement selected sequence 5 5 Translating sequences
278. t is checked the graph shows the GC content in the 3rd codon position only for each frame where frame 1 red frame 2 green and frame 3 blue Chromatogram This is available with chromatogram traces and displays the chromatogram trace above the sequence If Qual is checked the quality scores are displayed as a blue graph overlaid on the chromatogram For more information on viewing chromatograms see section 5 6 Stylized DNA Helix Shows the bases in your sequence as a rotating DNA helix To turn off the rotation uncheck the Animated option Protein sequence graphs Amino Acid Charge This runs the EMBOSS charge tool to plot a graph of the charges of the amino acids within a window of specified length as the window is moved along the sequence Hydrophobicity This displays the Hydrophobicity of the residue at every position or the aver age Hydrophobicity when there are multiple sequences pl pI stands for Isoelectric point and refers to the pH at which a molecule carries no net elec trical charge The pl plot displays the pl of the protein at every position along the sequence or the average pI when multiple sequences are being viewed 5 2 8 Advanced sequence view options Advanced options for controlling the look of sequences and alignments are under the Ad vanced tab These options are as follows Layout e Wrap sequence This wraps the sequences in the viewing area e Linear view on circular sequences This forces
279. t to general system backups safely since they are made when Geneious is in a non running state These backups can also be safely moved around including to other machines O ANOO Back Up Data Back Up O Export selected folder Local Can be re imported in to a database Archive all local data and settings Zip your entire data folder including settings Can only be loaded as an entire database Store backups on a separate hard drive if possible Cancel Figure 4 4 Using the backup tool 4 5 1 Restoring a backup e Geneious format file The easiest way to restore this is to drag and drop the Geneious file in to the folder in Geneious where you want it to go Alternatively you can use Restore Backup in the File menu and the backup will be added under the Local folder in your current database 60 CHAPTER 4 MANAGING YOUR LOCAL DOCUMENTS e Archive all data and settings It is strongly recommended that you use Restore Backup in the file menu to load the zip file rather than unzipping it manually Some operating systems may not be able to unzip the data correctly The Restore Backup command will unzip your backed up data directory to a folder of your choosing which you can then load immediately If you choose not to load it immediately you can switch to the restored data directory by going to Preferences in the Tools menu and changing the Data Storage Location on the General tab 4 6 Document History The histo
280. t will be imported as a blank sequence Reset to Defaults Cancel fox Figure 8 5 Selecting a reference sequence when importing BED GFF or VCF files For more information on these file formats see e BED format http genome ucsc edu FAQ FAQformat htmlitformat 1 e GFF format http www sanger ac uk resources software gff spec html e VCF format http samtools github io hts specs VCFv4 2 pdf Note that in BED format the first base is numbered 0 rather than 1 and Geneious accounts for this when it imports the file so your annotations will be shifted 1 bp to the right compared to their positions listed in the BED file 8 2 3 Transferring annotations from other sequences Copy to You can copy annotations from one sequence to other sequences in the same alignment or assembly document by right clicking on the annotation and choosing Annotation Copy to This will give you the option of either transferring the annotation to the consensus sequence reference sequence if there is one or any of the other sequences in the alignment or assembly You can also use the associated options Copy all x to to copy all annotations of the currently selected annotation type on the selected sequence and Copy all in selected region to to copy all annotations in the selected sequence In each case the annotation s will be copied across regardless of the similarity between the sequences Transfer Annotations Using Transfer Annota
281. tabase sequence that corresponds to a BLAST hit To retrieve the full sequence select a BLAST alignment and go to File Download Documents or click the Download Full Sequence s button located above the viewer tabs The full sequence will be available in the Sequence View tab once the download has completed and the region that matches the query sequence will be annotated as BLAST Hit see Figure 15 3 In addition the annotations from the full sequence will be transferred over to the BLAST alignment and can be viewed in Alignment view 15 3 NCBI BLAST 205 Sequence Annotations Alignment View Alignment Annotations Dotplot Dotplot Self Distances gt Yeo T OB E control region D loop 4 000 470 iy ND1 COS Pea d EN 4 ND2 CDS 4 COX2 CDS ATP8 CDS Aps COS ND6 CDS CYTB CDS Gz gt o gt ATP6 CDS Qi cox3 cos 2 0 a 5 0 Mouse over base 11 575 T Figure 15 3 Document After Full Sequence Download 15 2 2 Query centric view This view displays all of the hits to your query in a single alignment Results of single BLAST searches can be viewed in query centric view instead of a hit table by clicking the Query Cen tric View tab at the top of the document table Or you can choose to only return a query centric alignment when you set up the BLAST search This option is particularly useful for batch BLAST as only one alignment per query is returned and all the results are displayed in a sin
282. te pair _ Linker Sequence Y Allowupto 1 mismatches r M Trim End Adapter Primer Barcode Sequence CTGAGACTGCCAAGGCACACAGGGGATAGG M Allow up to 5 8 mismatches when finding near end of sequence M Find adapter primer barcode anywhere in sequence with up to 2 8 mismatches oo C Esa Figure 10 3 Options for separating reads by barcodes using a custom barcode set a fixed adaptor and a fixed end primer 132 CHAPTER 10 ASSEMBLY AND MAPPING check Specific barcode and enter the sequence Alternatively if you do not know your barcode sequences you can just enter the length of your barcodes and Geneious will automatically identify what the barcodes are Separate reads by barcodes only sorts by barcodes at the 5 end of the sequence but primers adapters or barcodes on the 3 end of the sequence can be trimmed off by checking Trim End Adaptor Primer Barcode Either enter a specific sequence to trim or add your 3 barcode to your custom barcode set and use the text END_BARCODE in the sequence box Primer trimming can also be performed after separating by barcodes using Trim Ends see section 10 1 1 For further information on splitting barcode data hover the mouse over any of the settings in the Separate Reads by Barcode options window 10 2 De novo assembly This can be used to assemble a small number of Sanger sequencing reads i e forward and re verse reads of the same sequence
283. ters for your organization can be saved as a profile and then shared on the Shared Database for everyone to use The operation settings button x appears in the bottom left corner of any options window Click on this button to reset to defaults load a profile save a new profile or manage your existing profiles as described below Saving a profile To create a profile set the options up the way you want click the operation settings button then choose Save Current Settings You can then enter a name for your profile and choose whether it is shared For a description of shared profiles see the section on sharing profiles When you save a profile it is attached to the particular analysis window that you have open Eg if you save a profile for Alignment it can only be loaded for Alignment not for Assembly Loading a profile To load a profile click the operation settings button and choose Load Profile and click on the name of the profile you want to load The settings for the operation will immediately update to reflect the profile Note Sometimes when you load a profile the settings may not exactly match what was saved 219 220 CHAPTER 18 SAVING OPERATION SETTINGS OPTION PROFILES This is because the available settings can change depending on what type of documents you have selected Managing profiles Click on Manage Profiles under Load Profile to see a list of profiles with options for delet ing editing importing and expo
284. th the Show Annotations box is a filter text field Typing a term in this field will highlight any annotations that contain the entered text in their name or qualifiers To filter for a term in a specific field click the down arrow to the right of the box and choose the field and term you wish to search for 8 1 VIEWING EDITING AND EXTRACTING ANNOTATIONS 97 To quickly find and move between instances of a particular annotation type on a sequence click the small left right buttons to the right of each annotation type This will move the selection in the sequence view to the next or previous instance of that annotation type This is useful for navigating large genomes or assemblies To customize the way an annotation type is displayed click on the preview of the annotation arrow in the Annotation and Tracks window This will bring up a popup menu containing the following options e Show only type annotations Turns off all other annotation types and shows only this type e Show type labels When this is unchecked only the annotation arrow is shown on the sequence and not the annotation name e type labels gt Allows you to customize which property of the annotation is displayed as the name e Show above sequence Moves the annotation so that it is sitting above the sequence rather than on or below it e Edit Color Allows you to change the color of the annotation arrow e Edit all type annotations Allows you to ch
285. the Allow Editing button enters edit mode and allows you to modify residues and gaps by typing them on the keyboard In an alignment you can quickly insert gaps by selecting a region of a sequence then clicking and dragging the selection You can also quickly select regions of alignments by triple and quadruple clicking a point When editing an alignment pressing 22 CHAPTER 2 THE GENEIOUS MAIN WINDOW 2 1 The Sources Panel The Sources Panel displays your stored documents and contains the services Geneious offers for storing and retrieving data The plus symbol indicates that a folder contains sub folders A minus indicates that the folder has been expanded showing its sub folders Click these symbols to expand or contract folders Geneious Sources Panel allows you to access e Your Local Documents e NCBI databases Gene Genome Nucleotide PopSet Protein Pubmed SNP Structure and Taxonomy An EMBL database Uniprot e Shared databases if set up e Your contacts Geneious databases if collaboration is installed All these services will be described in detail later in the manual You can view options for any selected service with the right mouse button or by clicking the Options button at the bottom of the Sources Panel in Mac OS X For more information on managing folders in the Sources panel see section 4 1 2 2 The Document Table The Document Table displays summaries of each document in a se
286. the Geneious toolbar to request help It is also possible to turn off the NCBI web services by editing this file this is not possible via the preferences inside Geneious 22 3 Specify license server location This can be specified by editing the geneious properties file in the installation directory see 22 2 2 Scroll down to the license server settings and change override property flexnet_server hos and override property flexnet_server port to the settings you require Remove the at the start of these lines for the setting to be used 22 4 DELETING BUILT IN PLUGINS 241 22 4 Deleting built in plugins Features of Geneious can be turned off in preferences as described in the section above on changing default preferences If you really want to delete a feature completely so your users can t reinstate it you should shut down Geneious go to the installation directory into the bundledP lugins directory and delete the desired plugin jar files folders 22 5 Max memory On Windows and Linux edit the files Geneious default 64bit vmoptions or 32bit as appropriate and Geneious in use vmoptions in the installation directory and change the Xmx value to your preferred setting On Mac OS X edit the Applications Geneious app Contents Info plist file and find the VMOpt ions section and modify the Xmx setting It is important on Mac OS X to ensure that this value is set appropriately after an upgrade because users can often find
287. the discovered junctions By default at least 2 reads must support the discovery of a junction in order for it to be used during the next pass This thresh old can be adjusted under More Options by changing the Minimum support for structural variant discovery setting Junctions used during the second mapping pass are annotated on the reference sequence under a track named after the reads Each junction annotation has the following properties e Junction Type This will be Deletion for deletions up to 1000 bp For longer deletions or structural variants this will be Rearrangement with inversion potentially appended e Intervals Each junction consists of two annotation intervals of a single nucleotide in 138 CHAPTER 10 ASSEMBLY AND MAPPING length which cover the final nucleotide used in the reference sequence before the read jumps and continues elsewhere For junctions of type Deletion the junction is represented as a single annotation with two linked intervals For junctions of type Rearrangement the junction site is split into two separate annotations each with a jagged edge on one side of the interval to indicate the side which jumps elsewhere Deletion Size This is present when Junction Type is Deletion Reads supporting discovery Indicates the number of reads that supported discovery of this junction during the first pass This may be lower than the advanced minimum support setting in cases where other reads supported discovery o
288. the setup click OK to start Tell me how to set it up myself a Setup Options Geneious is downloading the required files You may continue to use Geneious ncbi blast 2 2 28 universal macosx tar gz Downloaded 4 457 of 266 091 MB 1 67 Approximately 13 minutes 8 seconds remaining b Downloading Figure 15 4 Setting Up Custom BLAST If the file meets these requirements it will be added as a database otherwise you will be in formed of the problem Creating a database from local documents To create a BLAST database from sequences in your local documents folders first select the doc uments that you want Then go to Tools Add Remove Databases Add BLAST Database and select Custom BLAST from the Service drop down box Enter a name for the database and click OK 15 4 CUSTOM BLAST 209 Service Custom BLAST Database Name My Database C Use 1 selected sequences Contents e Create from file on disk Type _ Do not check file for duplicate names or invalid bases residues better performance a cancel OK Figure 15 5 Adding a FASTA database 15 4 4 Using Custom BLAST Once you have added one or more databases they will appear under Custom BLAST in the BLAST database drop down Figure 15 6 These can be used in exactly the same way as the NCBI BLAST ones 210 CHAPTER 15 BLAST COGs Not set up Click Add Remove Databases for setup NCBI Gen
289. then click OK to close the Plugins window To run LASTZ select the sequences or alignment you wish to align select the Align Assemble button from the Toolbar and choose Align Whole Genomes For more information on LASTZ and its options please refer to the original documentation for the program http www bx psu edu rsharris lastz 9 2 6 Translation alignment In a translation alignment nucleotide sequences are translated into protein the alignment is performed on the protein sequence and then translated back to nucleotide sequence Transla tion alignments can be performed with any of the alignment algorithms in Geneious such as the Geneious aligner MUSCLE or ClustalW In the translation alignment options you can set the genetic code and translation frame for the translation as well as the alignment algorithm you wish to use Under More Options you can set the parameters such as cost matrix gap open penalty and gap extension penalty for the alignment 9 2 7 Combining alignments and adding sequences to alignments Consensus Alignment allows you to align two or more alignments together and create a sin gle alignment and align a new sequence in to an existing alignment Select the sequences or alignment you wish to align and select the Align Assemble button from the Toolbar and choose Multiple Alignment Consensus alignment allows you to choose which alignment algorithm to use for aligning the consensus sequences All of the pairwise a
290. these criteria are combined Match Any requires a match of one or more of your search criteria This is a broad search and results in more matches Match All requires a match all of your search criteria This is a narrow search and results in fewer matches 56 CHAPTER 4 MANAGING YOUR LOCAL DOCUMENTS Match all BS of the following Author E contains 3 Drummond AJ dp Date published ES is between orsanz003 and 3 31 Dec 2005 q Create Agent Search Fewer Options Name Summary R Choosing appropriate substitution models for the phylogenetic analysis of protein coding seque Beth Shapiro Andrew Rambaut amp Alexei J Drummond 2005 Mol Biol Evol 23 7 9 Tree measures and the number of segregating sites in time structured population samples Roald Forsberg Alexei J Drummond amp Jotun Hein 2004 BMC Genet 6 35 Molecular phylogeny of coleoid cephalopods Mollusca Cephalopoda using a multigene approach 4 effect of data partitioning on resolving phylogenies in a Bayesian framework a ja Q Choosing appropriate substitu 2D Tree measures and the numb 2 Molecular phylogeny of coleoi Figure 4 3 Advanced Search 4 2 2 Similarity BLAST like searching It is possible to search your local documents not only for text occurrences but by similarity to sequence fragments Click the small arrow at the bottom of the large T to the left of the search dialog
291. this option is not selected then each interval in the annotation will be extracted to a separate sequence and grouped into a list Bulk extraction Bulk extraction of annotations can be done in two ways 1 Select all the annotations you want to extract either on the sequence or in the Annotations table and click Extract As with multi interval annotations you are given the option to concatenate all the annotations into a single sequence 2 Go to Extract Annotations under the Tools menu Using this interface all annotations on the selected sequences which match certain criteria e g a particular annotation type or gene name can be extracted in bulk without needing to select the annotations on the sequence first To define what annotations to extract select the value of the annotation type or property qualifier that you want to extract by in the chooser see Figure 8 4 To set more than one criteria click the button to add an additional row of options and choose to either Match all or Match any of the criteria ene Extract Annotations Annotation type of Is Coverage High Gi What to extract Intersecting Annotations Exclude Concatenate regions within each sequence Include Intergenic Regions y Extraction Context Include upstream 5 flanking bases Include downstream 3 flanking bases Cancel o as Figure 8 4 The Extract Annotations Interface 8 2 ADDING ANNOTATIONS 101 What to Extract
292. tion on these algorithms Pairwise alignment in Geneious Like a dot plot a pairwise alignment is comparison between two sequences with the aim of identifying which regions of two sequences are related by common ancestry and which regions of the sequences have been subjected to insertions deletions and substitutions To run a pairwise alignment in Geneious select the two sequences you wish to align and choose Align Assemble Pairwise align The options available for the alignment cost matrix will depend on the kind of sequence e Protein sequences have a choice of PAM and BLOSUM matrices e Nucleotide sequences have choices for a pair of match mismatch costs Some scores distinguish between two types of mismatches transition and transversion Transitions A gt G C T generally occur more frequently than transversions Differences in the ratio of transversions and transversions result in various models of substitution When applicable Geneious indicates the target sequence similarity for the alignment scores i e the amount of similarity between the sequences for which those scores are optimal Both protein and nucleotide pairwise alignments have choices for gap open gap exten sion penalties costs Unlike many alignment programs these values are not restricted to integers in Geneious 9 2 SEQUENCE ALIGNMENTS 113 e00 Pairwise Multiple Align Geneious Alignment MUSCLE Alignment Realign Region Translation A
293. tions you can copy annotations to the reference and or consensus se quence of an alignment or assembly This function can be accessed from the Annotate and 8 2 ADDING ANNOTATIONS 103 Predict menu or in the Live Annotate and Predict tab in the sequence viewer To set a se quence as a reference sequence Ctrl click on it and choose Set as reference sequence Annotations will only be transferred where the annotated sequence and the reference consensus sequence have at least the specified minimum similarity All transferred annotations will be annotated with a Transferred From qualifier indicating the names of the sequences the an notation came from sorted in order of decreasing similarity and a Transferred Similarity qualifier which indicates the percentage similarity of the most similar sequence the annotation was transferred from Annotations of the same type and covering the same interval that would be transferred from multiple sequences are merged together such that the name of the transferred annotation will consist of the names of all contributing annotations sorted in order of decreasing similarity Similarly if contributing annotations have different qualifier values the resulting qualifier value will consist of all contributing qualifier values sorted in order of decreasing similarity The percentage similarity is the sum of the similarity values for each position as a fraction of the sum of the maximum similarity values of the base
294. tivate their license on the machine The admin should not activate licenses for users as this will prevent the user from activating the license themselves 1 4 3 Borrow Floating License This item is only available to users for a floating license administered through a FLEXnet li cense server Borrowing a license allows you to borrow one of the seats of a floating license so you can use it even when disconnected from the network Since this decreases the number of seats available for other users borrowing can only occur with the authorization of the sys tem administrator If your borrowing is approved the system administrator will provide you with a borrow file authorizing the borrow To borrow a license check Borrow in the menu 20 CHAPTER 1 GETTING STARTED and navigate to this file when prompted by Geneious Borrowed licenses have an expiry date when they will automatically be returned to the server but if you are finished with the license before the expiry date please uncheck Borrow in the menu while connected to the network in which the license server resides so that the license is returned to the server and is available to other users again 1 4 4 Release License Personal licenses can only be activated on a maximum of three computers at one time If you no longer need to have Geneious available on a computer where you have activated it you can release the license so it is available for use on another computer Lic
295. to activate a license download plugins and search external databases like NCBI Geneious needs to be able to connect to the internet If you have a firewall preventing di rect access or are behind a proxy server you may need to manually configure your connection settings You can do this as follows 1 Check the browser you are using These instructions are for Explorer Safari Chrome and Firefox 2 Open your default browser 3 Use the steps in Figure 1 3 for each browser to find the connection settings 4 Now in Geneious go to Tools Preferences and click on the General tab There are five options in the drop down options under Connection settings Figure 1 2 1 2 GENEIOUS SETUP 15 e Use direct connection Use this setting when no proxy settings are required Use browser connection settings This allows Geneious to automatically import the proxy settings This may not work with all web browsers e Use HTTP proxy server This enables two text fields Proxy host and Proxy port This information is in your browser s connection settings Use this if your proxy server is an HTTP proxy server Please see step 3 e Use SOCKS proxy server Autodetect Type This enables two text fields Proxy host and Proxy port This information is in your browsers connection settings Use this if your proxy server is a SOCKS proxy server Please see step 3 e Use auto config file This enables one text field called Config file location
296. ts Results are displayed as an annotation track on the reference sequence By default annotations are colored based on the TPM property ranging from blue for 0 through to white for the mean TPM up to red for the highest TPM for any gene in the sample In the results view by clicking on the little down arrow to the left of the track s name you can choose to color by a different property The values for RPKM FPKM and TPM as well as the raw read counts are entered as proper ties on the annotation and can be displayed by mousing over an annotation To export these values as a table switch to the Annotations tab above the sequence viewer then click the Track button and choose the Expression track to display Then click the Columns button and add the columns for FPKM RPKM TPM and or the raw counts Once you have the columns you need you can export the table in csv format by clicking Export table 11 2 2 Comparing Expression Levels Geneious is able to find differentially expressed genes between two samples Comparison be tween more than two samples is not currently supported 150 CHAPTER 11 ANALYSIS OF ASSEMBLIES AND ALIGNMENTS To compare expression levels you must first run Calculate Expression Levels from the Anno tate amp Predict menu on each contig assembly of the two datasets These assemblies must use the same reference sequence This will save an Expression Level track for each contig on the reference sequence To compar
297. u can also check for off target sites with bulges and deletions by allowing some of these mismatches to be indels Maximum mismatches allowed to be indels controls this Note that increasing these numbers will increase the complexity of the search and therefore the time it takes to complete To help speed up off target analysis Geneious can filter out CRISPR sites with high identity off targets before performing the more intensive search for off targets with mismatches There are three Strategy options to for this e Slowest Score all sites No initial filtering for high identity off target sites scores will be calculated for all matching off targets e Normal Discard worst sites Filters out CRISPR sites with off targets that exactly match the 12 bp immediately adjacent to the PAM site e Fast Discard more sites Filters out CRISPR sites with off targets that exactly match the 8 bp immediately adjacent to the PAM site 198 CHAPTER 14 CLONING Pair CRISPR sites To help minimize off target interactions the mutant Cas9 D10A Nickase can be used with a pair of gRNAs on complementary strands of a target sequence This induces a single stranded break at each site simulating a double stranded break overall Off target interactions of any one gRNA target will then be only a single nick which is repaired with high fidelity This process is described by Zhang et al 2013 By selecting Pair CRISPR sites only CRISPR sites that are within
298. uences contain type IIS restriction sites that you do not want be involved in the as sembly then you will need to engineer each site out of your fragment using PCR taking care to avoid altering any gene product sequences e Removal of unused primer annotations If one or more of your sequences contain primer annotations that do not define a boundary for assembly then you should remove these annotations If you do not remove these annotations then Geneious may interpret them as boundaries of your sequence which will result in truncated inserts and incorrect as sembly 14 7 TOPO Cloning TOPO Cloning lets you ligate a single fragment into a Vector within only 5 minutes using the natural activity of Topoisomerase I which recognizes a specific motif 5 C T CCTT 3 on the DNA TOPO is a registered trademark of Invitrogen Corporation The TOPO cloning function under the Cloning menu allows you to insert linear fragments into either linear TOPO vectors when a TOPO site is present at the extremities or into circular TOPO vectors You can select as many sequences at once as you like they will be ligated into each other in a batch operation e Three different options TA Blunt or Directional cloning are shown on the top If Direc tional is selected the user can define an overlap sequence If this field is blank it has the same effect as Blunt cloning e The field below shows which of the selected sequences have been detected as vectors al
299. umber of example Workflows for a variety of tasks that you can try Workflows can be run or managed via the Tools menu or if configured via the Geneious Toolbar See section 16 1 Workflows can be shared with other people either by exporting and importing them or if you are connected to a shared database by ticking the option to share them See Figure 16 1 If you have any programming knowledge you can even add customised code in Java See section 16 3 16 1 Managing Workflows Workflows can be accessed from the Workflows menu under the Tools The Workflows menu allows you to select and run saved Workflows manage your Workflows and create new ones If you are going to be using Workflows routinely then for quick access you can add Workflows to the main Geneious Toolbar To do this right click on the main Geneious Toolbar choose Customize and check Workflows The Manage Workflows window lists all available workflows and contains options for view ing editing copying deleting exporting and importing workflows Each Workflow listed in the Manage Workflows window with an icon next to it will be shown in the drop down un der the main Workflows menu See Figure 16 1 The order in which Workflows appear in the drop down menu can be controlled using the Move Up and Move Down buttons see Figure 16 1 Multiple workflows can be selected using Shift or CNTRL click and they can then be exported or added removed from the drop down list in
300. umn values can be copied This is a quick method of extracting searchable infor mation such as an accession number To copy a value right click Ctrl click on Mac OS X on it and choose the Copy name option where name is the column name Sorting All columns can be alphabetically numerically or chronologically sorted depending on the data type To sort by a given column click on its header If you have different types of documents in the same folder click on the Icon column to sort then according to their type Managing Columns You can reorder the columns to suit Click on the column header and drag it to the desired horizontal position You can also choose which columns you want to be visible by right clicking Ctrl click on Mac OS X on any column header or by clicking the small header button in the top right corner of the table This gives a popup menu with a list of all the available columns Clicking on a column will show hide it Your preference is remembered so if you hide a column it will 24 CHAPTER 2 THE GENEIOUS MAIN WINDOW remain hidden in all areas of the program until you show it again As well as items to show hide any of the available columns there are a few more options in this popup menu to help you manage columns in Geneious Lock Columns locks the state of the columns in the current table so that Geneious will never modify the way the columns are set up You can still change the columns yourself howev
301. upe setting hover the mouse over the setting or click the help question mark button next to the custom options under More Options 44 Batch Rename Batch rename is located under the Edit menu and can be used to edit any field in multiple documents at once It can also be used to batch edit any property of sequences within an alignment or sequence list Existing fields can be replaced with a combination of values from other fields e g Name re placed with Organism and or Accession and fixed text can be added to the beginning or end of existing fields The advanced options under the More Options button enables the use of regular expressions to replace a specific part of one field with another property or text string Click the Help button in this section for more information on formatting expressions 4 5 Backing up your local documents It is important to keep frequent back ups of your data because computers can fail suddenly and unexpectedly A computer can be replaced but your data is much harder to replace The best way to back up all of your data and settings in Geneious is to use the Back Up button in the toolbar or select Back Up Data in the File menu Figure 4 4 Note Due to the way the local database works it is important that Geneious is not accessing the database when a backup is taken For example Mac users with Time Machine will have backups taken during the day but if Geneious is running when those backups are taken
302. us 9 0 Data folder inside the directory you specify 22 2 Change default preferences 22 2 1 Change preferences within Geneious Start a fresh copy of Geneious set it up the way you want Shut down and then copy Geneious 9 0 Data user preferences xml to the Geneious install directory e g C Program 239 240 CHAPTER 22 ADVANCED ADMINISTRATION Files Geneious on Windows XP and rename it to default_user_preferences xml Now when users start Geneious for the first time they will get the configuration you set rather than the normal default Examples of features you can change e Turn off automatic updates e Set default custom BLAST location e Set up a shared Database e Set up a proxy server default e Turn off particular plugins Any users who have already run Geneious should click the Reset All Preferences button in the Geneious Preferences to load these defaults 22 2 2 geneious properties file Any preferences which can be set within Geneious can also be set from the geneious properties file which can be found in the Geneious installation directory On MacOS this file is located in Geneious app Contents Java R9 and above or Geneious app Contents Resources app R8 and earlier Some examples are present in the file already remove the hashes from the start of the lines and modify the values to use them If you need to find out how to set other preferences using this file please use the Support button in
303. ust connect Username Password Confirm password Email address optional C Connect every time I run Geneious 2 More Options Figure 19 1 Add New Account dialog box Choose a username and password now Enter your password twice for a new account You can also optionally add an email address Biomatters will need this if you require support regarding e g reset of password or deletion of accounts More Options You can change some of the defaults for new and exiting accounts 19 1 MANAGING YOUR ACCOUNTS 223 Account Name is the name displayed in the Services Panel for this account It defaults to your username if nothing is entered e Server is the server your account connects to default talk geneious com e Jabber Service Name is required by some other Jabber service providers such as Google Talk Don t enter anything here unless you know what you are doing e Port Number for Jabber servers running on a non standard port default 5222 gt Add New Account Create a new account on the server This account already exists just connect Username Password Confirm password Email address optional C Connect every time I run Geneious Account Name Server talk geneious com Jabber Service Name Port Number 5222 lt gt Fewer Options Figure 19 2 Add New Account dialog box with More Options 19 1
304. ve the results 6 Both the Geneious de novo and reference assemblers use a deterministic method even when spreading the work cross multiple CPUs such that if you rerun the assembler using the same settings and same input data it will always produce the same results 10 3 Map to reference Assembling to reference is used when you have known sequence and you wish to compare a number of reads of the same sequence with it to locate differences or SNPs To perform assembly to a reference sequence select the sequences and the reference sequence and click Align Assemble and choose Map to Reference Choose the name of the sequence you wish to use as the reference in the Reference Sequence chooser and click OK One contig will be produced per reference and this will display the reference sequence at the top of the alignment view with all other sequences below it The options available in the Map to Reference setup dialog are similar to those for de novo assembly see section 10 2 with a few differences detailed in the subsequent sections In the Methods panel you can choose between the standard Geneious assembler Geneious for RNAseq R9 or the BBMap Bowtie or Tophat mappers if you have these plugins installed See chapter 11 for details on identifying differences or SNPs in your assembly 10 3 1 Choosing reference sequences In version 8 1 and above it is not necessary to select the reference sequence prior to choosing Map to Reference Inste
305. ved these may be fixed or configurable For example with the sequence viewer if wrapping is on you are able to choose the width at which the sequence is wrapped but if wrapping is off both the width and height will be fixed Format Controls image format Vector formats PDF SVG and EMP are ideal for publication because they won t become pixelated Raster formats PNG and JPG are easier to share great for emailing and posting on the web If you wish to edit the file outside of Geneious SVG or EMF format should be used SVG files can be edited in tools such as Adobe Illustrator or Inkscape and EMF files can be edited on Windows using PowerPoint or LibreOffice Draw on Mac or Linux the Mac version of PowerPoint can t modify EMF files With SVG or EMF it is possible to ungroup components of the graphic for editing and because they are vector graphics they will scale without becoming pixelated Resolution Only applies to raster formats PNG and JPG and is used to increase the number of pixels in the saved image We recommend increasing the resolution to at least 300 for printing PNG or JPG files Chapter 4 Managing your Local Documents 4 1 Organizing your local documents Geneious documents are stored in a hierarchal arrangement of folders under the Local folder in the Sources Panel Clicking on a folder will display its contents in the Document Table Next to each folder name in the hierarchy is the number of documents it contains
306. volving from one base to another in a given period of time This section briefly discusses some of the substitution models available in Geneious Most models are variations of two sets of parameters the equilibrium frequencies and relative substitution rates Equilibrium frequencies refer to the background probability of each of the four bases A C G T in the DNA sequences This is represented as a vector of four probabilities m4 nC TG TT that sum to 1 Relative substitution rates define the rate at which each of the transitions A gt G C T and transversions A C A e T C gt G G e T occur in an evolving sequence It is represented as a 4x4 matrix with rates for substitutions from every base to every other base Additionally gaps are not penalized when using the Geneious Tree Builder Comparisons in volving any gaps are ignored when calculating the distance matrix Jukes Cantor This is the simplest substitution model It assumes that all bases have the same equilibrium base frequency i e each nucleotide base occurs with a frequency of 25 in DNA sequences and each amino acid occurs with a frequency of 5 in protein sequences This model also assumes that all nucleotide substitutions occur at equal rates and all amino acid replacements occur at equal rates see Jukes and Cantor 1969 HKY The HKY model assumes every base has a different equilibrium base frequency and also as sumes that transitions evolve at a diff
307. w what the relative orientation or expected distance is between the reads you should ask your sequencing data provider When you click OK if you chose to pair by parallel lists of sequences Geneious will create a new document containing the paired reads If you chose to pair an interlaced list of sequences or modify settings for some already paired data Geneious will just modify the existing list of sequences to mark it as paired If you choose to split reads based on the presence of a linker sequence e g for 454 data the original sequences will be unmodified and the split reads will be created in a new document The default behaviour is to ignore sequences shorter than 4 bp either side of the linker but this can be customized from the Edit Linkers option in the paired reads options Polonator sequencing machine reads can be split using the Split each read in half option 10 1 5 Merging paired reads If paired reads were sequenced with an insert size shorter than twice the read length then pairs may overlap with each other in which case it can be useful merge to each pair into a single longer read After setting up paired reads see section 10 1 4 use Merge Paired Reads from the Sequence menu to merge them This function uses BBMerge from the BBtools suite For a detailed explanation of any BBMerge setting hover the mouse over the setting or click the help question mark button next to the custom options under More Options
308. will produce one or more output documents all of which are grouped together and used as input into the next Step in the Workflow However it is possible to create Workflows where each Step in the Workflow may be invoked in parallel on different sets of documents For example if the first Step in the Workflow uses the For Each Sequence Extract Sequences From List Step then each input sequence is placed into a separate document and the following Step will be invoked independently on each sequence Each call to the following Step may produce one or more documents Each of these sets of out put documents are used independently as inputs to multiple invocations of the next Workflow Step Alternatively you could group these results together again using Group sequences or Group Documents to use as input to a single call to the next Workflow Step No matter what document grouping or separation For Each Operations are used each Step in the Workflow is always run to completion on all data sets before starting on the next Step in the Workflow 216 CHAPTER 16 WORKFLOWS Inputting documents into later stages of a Workflow It is also possible to insert documents into later stages of the workflow either from additional documents the user selects in the options when they first start the workflow using the Add document chosen when running workflow Step or to use documents generated from earlier stages in the workflow using the Combine With Earlier Do
309. xpressed genes from the sample are calculated and the median values of these from each sample are used to normalize For example if one sample has a median twice as high as the other sample then the counts for each gene need to be halved to make them comparable with the other sample Total Count Excluding Upper Quartile The expression level of all expressed genes from the sample are calculated and the total number of reads fragments or transcripts from the lowest 75 of those are totaled Values are normalized between samples based on this total e Median of Gene Expression Ratios For each gene the ratio of the expression level be tween samples is calculated Then the median ratio across all expressed genes is used as 11 2 ANALYZING EXPRESSION LEVELS 151 the normalization scale This normalization method is the same as that implemented by DESeq All of these normalization methods and more are described and compared by Dillies et al 2012 and they recommend using Median of Gene Expression Ratios rather than the other three normalization methods implemented here One reason for this is that a few highly ex pressed genes can greatly affect the total number of transcripts produced so this can distort the fraction of the total reads that contribute to genes with lower expression The choice of normalization method determines the Differential Expression Ratio for each gene P Value Calculation In addition to calculating the dif
310. y clicking the Mismatch Options button Click the OK button and testing will commence Once complete a dialog will present the re sults This dialog tells you how many of the sequences were compatible with the specified primers and probes and provides details and choices very similar to the one described in sec tion 13 1 10 The compatible primers can be annotated onto the sequences in a similar manner to that when designing primers Additionally if the primer sequences were not already anno tated with a primer annotation they will be annotated during testing 13 6 Characteristics for Selection The Characteristics for Selection option will determine the primer characteristics of a selection of sequence within a larger sequence Select a region of 36bp or less in the Sequence View and choose Primers Characteristics for Selection The primer characteristics will then be added as an annotation over the exact region that was selected This will also work on multiple selected regions in the Sequence View Hold the Ctrl key while clicking and dragging to select multiple regions simultaneously 13 7 Convert to Oligo Geneious can convert nucleotide sequences into primers This is necessary for sequences to show up in the oligo database If your sequences are less than 36 bp long this operation will also determine the primer characteristics of the sequences such as melting point To do this select your sequences and choose Primers Convert to O
311. you choose this Geneious will prompt you to log in The rest of the options are the same as for any local job and the job will progress in the same way as if run locally only using the remote resources provided by the server If the job is likely to complete quickly you should just run it locally but if it requires a lot of memory more than your local machine has for instance or if it will take a long time to process you should choose to run it on the server You can check the status of your job in the operations table in Geneious You can also shut Geneious down once your job has been submitted to the server and if the job has completed when you log back in you ll be able to retrieve your results If your jobs were running when you shut down Geneious will request progress from the server when you restart and either show you your completed jobs or show you the progress dialogue so you can see how far along the job has gone Figure 21 4 236 CHAPTER 21 GENEIOUS SERVER A Geneious Alignment A MUSCLE Alignment ClustalW Alignment Realign Region Translation Align A MAFFT Alignment 4 Consensus Align A Profile Align O mauve Genome LASTZ Alignment Run On my computer on Geneious Server 65 similarity 5 0 4 0 E 8 Global alignment with free end gaps Cost Matrix Gap open penalty 12 Gap extension penalty 3 M Automatically determine sequences direction Alignment type Bui
312. your local documents meta data types and pro gram preferences A file in Geneious format will usually have a geneious extension or a xml extension This format is useful for sharing documents with other Geneious users and backing up your Geneious data Geneious tutorial This is an archive containing a whole bundle of files which together comprise a Geneious ed ucation document This format can be used to create assignments for your students bioinfor matics tutorials and much more See chapter 17 for information on how to create such files GFF annotations The GFF format contains sequence annotation information and optional sequences You can use a GFF file to annotate existing sequences in your local database import entirely new se quences or import the annotations onto blank sequences MEGA alignment The MEGA format is used by MEGA Molecular Evolutionary Genetics Analysis Molecular structure Geneious imports a range of molecular structure formats These formats support showing the locations of the atoms in a molecule in 3D PDB format files from the Research Collaboratory for Structural Bioinformatics RCSB Protein Database e mol format files produced by MDL Information Systems Inc e xyz format files produced by XMol e cml format files in Chemical Markup Language e gpr format ghemical files e hin format files produced by HyperChem e nwo format files produced by NWChem 38 CHAPTER 3 IMPORTING A
313. ypes Document type Geneious Icon Nucleotide sequence Oligo sequences Enzyme Sets Chromatogram Contig Protein sequence Phylogenetic tree 3D structure xl RAINERIA Y Sequence alignment Journal articles y PDF Other documents dragged documents from one local folder to another this action will move the documents so that a copy of the document is not left in the original location In external databases such as NCBI the documents will be copied leaving one in its original location Drag and copy While dragging a document over to your folder hold the Ctrl key Alt Option key on Mac OS X down This places a copy of the document in the target folder while leaving a copy in the original location This is useful if you want copies in different folders Folders themselves can also be dragged and dropped to move them but they cannot be copied The Edit menu Select the document and then open the Edit menu on the menu bar Click on Cut Ctrl X command X or Copy Ctrl C command C Select the destination folder and Paste Ctrl V command V the document into it 4 2 SEARCHING AND FILTERING LOCAL DOCUMENTS 53 4 1 2 Deleting Data and the Deleted Items folder The Deleted Items folder is located underneath the local document folders in the Sources panel When a folder or document is deleted Geneious moves the data to the Deleted Items folder instead of erasing it immediately This means the data can be recovered i

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