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Geneious User Manual

1. in that alignment column 3 5 1 Pairwise sequence alignments There are two types of pairwise alignments local and global alignments A Local Alignment A local alignment is an alignment of two sub regions of a pair of sequences 21 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 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 16 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
2. 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 reguiring more pages Size Controls the size the printed region on the paper Effectively increasing the size reduces the margins on the page 2 10 2 Saving Images Choose save to image file from the file menu The following options are available Size Controls the size of the image to be saved Depending on the document view being saved these may be fixed or configurable For example with the seguence viewer if wrapping is on you are able to choose the width at which the seguence is wrapped but if wrapping is off both the width and height will be fixed Format Controls image format Vector formats PDF and SVG have the advantage over raster formats PNG and JPG that they don t become pixelly when magnified Vector formats are only available in the pro version Resolution Only applies to raster formats PNG and JPG and is used to increase the number of pixels in the saved image 52 CHAPTER 2 RETRIEVING AND STORING DATA Chapter 3 Analysing Data By the end of this chapter you should e Know about the main document viewers in Geneious e Understand the basic principles of bioinformatics e Be able to perform simple bioinformatics analyses with Geneious 3 1 Document Viewers
3. 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 2 1 5 The Toolbar The toolbar contains several icons that provide shortcuts to common functions in Geneious You can alter the contents of the toolbar to suit your own needs The icons can be displayed small or large and with or without their labels The Help icon is always available 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 The toolbar can be customized by right clicking Ctrl Click on MacOS on it This gives a popup menu with the following options e Show Labels Turn the text labels on or off e Large Icons Switch between large and small icons e A list of all available toolbar buttons Selecting deselecting buttons will show hide the buttons in the toolbar 2 1 6 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 20 CHAPTER 2 RETRIEVING AND STORING DATA 2 1 7 T
4. Confirm password Email address optional C Connect every time I run Geneious Account Name Server talk geneious com Jabber Service Name Port Number 5222 A Fewer Options Figure 9 2 Add New Account dialog box with More Options 116 CHAPTER 9 COLLABORATION PRO ONLY 9 1 2 Edit Account Details This option from the Collaboration menu or your account s context menu allows you to change the configuration you made when creating the account If you change your password Geneious will attempt to change it on the server the next time you connect For this purpose Geneious internally remembers your previous password as well so that it can still connect if you have entered your new password while disconnected 9 1 3 Connect Disconnect As all other collaboration related commands options for connecting to or disconnecting from your account are available both in the Collaboration menu and your account s context meu right click or on Ctrl click on MacOS on your account 9 1 4 Delete Account This option deletes your account configuration from Geneious Currently there is no option for deleting an account on the server 9 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 ap
5. The Entrez Popset database This database contains sets of aligned sequences that are the result of population phylogenetic or mutation studies These alignments usually describe evolution and population variation The PopSet database contains both nucleotide and protein sequence 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 Entrez Structure database This is NCBI s structure database and is also called MMDB Molecular Modeling Database It contains three dimensional biomolecular experimentally or programmatically determined structures obtained from the Protein Data Bank 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 2 4 PUBLIC DATABASES 35 Entrez Taxonomy This database contains the names of all organisms that are represented
6. allows you to enter how many megabytes of your com puters memory you wish to allow Geneious to use Search History This clears all the previously searched words in Geneious After this the auto completion drop box will be empty Connection settings These are described in the troubleshooting section of the manual 2 9 2 Plugins The Plugins tab Figure 2 14 contains a table of the currently available plugins for Geneious To enable a plugin select the checkbox next to it To disable a plugin deselect the checkbox next to it 2 9 3 Appearance and Behaviour Here you can change the way Geneious looks and the way it interacts with you 50 808 CHAPTER 2 RETRIEVING AND STORING DATA Preferences Plugins are optional modules that are not required in order for Geneious to function Plugins may be created by third party software vendors to provide additional features Use this page to enable disable plugins M 3 D structure importer and exp Chromatogram importer and vi Y Y Lu O m m Y v m m v M m Y M m Y a m S K m Y E m Mm Plugin Name 4 Clustal W importer ClustalW Collaboration beta DNA Strider importer Document Source Viewer Dotplot View EMBL database Endnote FASTA Importer exporter Generate consensus seguence Graphical Seguence View Graphical Tree View JMol MEGA exporter NCBI database New Seguence Newick importer Nexus importer Open reading
7. and Pro For details on the menu bar see section 2 1 7 1 2 7 Popup Menus Many actions can be guickly accessed for data items services and sometimes selections in a viewer via popup menus also known as context menus To invoke a popup menu for an item a v Show Labels v Large Icons h h h Li Li 7 Li 63 11 simply right click ctrl click on MacOS The popup menu will contain the actions which are relevant to the item you clicked 1 3 Troubleshooting 1 3 1 Geneious won t start Geneious has some minimum system reguirements It is compatible with the three most com mon operating systems Windows Mac and Linux Check that you have one of the following OS versions before you launch Geneious Geneious also needs Java 1 5 to run If you do not have this on your system already please 12 CHAPTER 1 GETTING STARTED Operating System System requirements Windows 2000 XP Mac OS X 10 4 Unix Linux download a version of Geneious that includes Java 1 5 from http www geneious com This involves downloading a larger file If you are a Mac user and have OS X 10 4 Tiger you will have to download Java 1 5 from http www apple com downloads macosx apple macosx updates javaformacosx104relea html 1 3 2 I get a connection error when trying to search using NCBI or EMBL If the message reads Check your connection settings there is a problem with your Internet connection Make
8. 8 759 Pair Primer Dimer 3 00 Pair Tm Diff 0 1 Figure 3 14 Primer design output 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 seguences then a dialog is shown describing how many had no matches and for what reasons To see why no primers or DNA probes were found for particular seguences click the Details button at the bottom of the dialog The dialog will then open out to display a list of all the seguences for which no primers or DNA probes were found For each of the seguences the following information is listed e Which of Forward Primer Reverse Primer Primer Pair and or DNA Probe could not be found in the seguence 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 3 7 2 Test Primers Primers and probes can also be quickly tested against large numbers of sequences to see which ones the primers will bind to By default this will only find sequences that match the primers exactly To test primers first select the primer or DNA probe sequences you wish to test or sequences which contain the de
9. Figure 3 4 A view of dotplot of two seguences in Geneious 3 1 DOCUMENT VIEWERS IN GENEIOUS 63 3 1 3 3D structure viewer Used to view molecular structures in 3D 3D structures can be obtained by searching NCBI s Structure database from within Geneious or by importing a range of 3D structure file formats such as e g PDB files from your hard drive To rotate the molecule and view it from an other angle drag the mouse while holding the left mouse button To zoom in and out press Shift mouse button and drag the mouse up and down or use the scroll wheel on your mouse Under Windows and Linux you can also move the molecule parallel to the viewing plane while pressing Ctrl Alt left mouse button This is currently not possible under Mac OS 3 D structure Sequence View Text View Summary Source Source v Position T Spin JP Center on click Reset position v IV Show Atoms Atom size 30 Y Show hydrogens 7 Show atom symbols Y Bons Y Show bonds Size Y Show H bonds Size f JY Show Backbone o fe Backbone type ribbons y 170 pi J a Backbone size y n i v J Measure Distances M hi y Left click on two atoms we to add a distance measurement E ae he i gt rement units Jone y Y Colors Atoms Default v Backbone Default v Y Visibility Filter Atoms Show All To rotate hold down the mouse button and drag To zoom hold down the shift key and mouse button and drag T
10. iw Figure 2 5 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 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 2 3 2 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 2 4 Public databases Geneious allows you to search several public databases in the same way that you can search your local documents The search process is described in section 2 3 2 4 PUBLIC DATABASES 33 15 Match all BS of the following Author HJ contains i Drummond AJ dh Date published P is between 1 TF o1jan2003 and 5331Dec2005 Create Agent Search Fewer Options Name Summary 2 Choosing appropriate substitution models
11. start downloading it again from the beginning 4 1 3 Adding Databases Now that you have set up the executables it is time to add databases to your BLAST Adding FASTA databases 1 Add Database Geneious can create a BLAST database from a FASTA file Select a FASTA file and choose whether it is a Protein or a Nucleotide database Select a FASTA File Nucleotide Database Protein Database Figure 4 1 Adding a FASTA database To create a database from the seguences in a FASTA file click on Custom BLAST in the Services Panel and click Add Database Navigate to the FASTA file that contains the seguences you want to BLAST and click OK There are two reguirements for a FASTA file to be suitable for creating a database from e The FASTA file must contain only the same types of seguence i e Nucleotide or Amino Acid e The sequences in the FASTA file must all have unique names If the file meets these reguirements 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 seguences in your local documents folders first select the documents that you want Then select Create BLAST Database in the Tools menu enter a name for the database and click OK 4 1 SETTING UP 101 4 1 4 Using Custom BLAST Once you have added one or more databases the applicable BLAST programs will appear under Custom BLAST in yo
12. 1793 1797 forward strand GAATTC ow GG GG HS S reverse strand CTTAAG ep la fey fe le Le l l vector index 3202 3206 3240 3244 C Keep fragments which are not part of the product Figure 10 3 Insert into Vector options dialog 10 3 INSERT INTO VECTOR 127 10 3 1 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 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 10 3 2 Vector Options e Polylinker region to cut within These options let you choose what region within the vector seguence to look for enzymes to cut within Geneious will examine the vector seguence for enzymes that have cut sites within this region and none outside it You can specify the polylinker in the following ways Annotation If the vector has one or more polylinker annotations annotated on it you can choose to use the interval covered by one such polylinker annotation directly 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 whic
13. 3 Choose the options you reguire 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 94 CHAPTER 3 ANALYSING DATA 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 selected 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 assembly as normal Click More Options in the assembly options to display the Alignment parameters Here you can change the parameters used by Geneious when aligning fragments together For sequences which are lower quality or contain many errors the gap penalty should be decreased and the mismatch score should be increased The algorithm Sequence assembly in Geneious uses a simple greedy algorithm which is very similar to that used in its multiple sequence alignment 1 Determine all pairwise distances using BLAST like search 2 Reject pairs who s hit is not at least as long as the minimum overlap length 3 Progressively align using Needleman Wunsch 16 the highest scoring pairs reverse complementing if necessary appending sequences to contigs
14. 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 each of the seguences will have the designed primers and probes added to them as seguence annota tions The annotations will be labelled with their rank compared to the other primers eg 1st 2nd where 1st is the best and what type they are Forward primer Reverse primer or DNA 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 an annotation The information will be presented in a popup box Alternatively double clicking on an annotation will display its details in the annotation editing dialog The best way to save a primer or DNA probe for further testing or use is to select the annotation for that primer and click the Extract button in the seguence viewer This will generate a separate short seguence document which just contains the primer seguence and the annotation so it retains all the information on the primer In the case of the reverse primer it should be reverse complemented When the Extract button is chosen for the reverse primer it will offer to reverse complement because the annotation runs in the reverse direction Choose Yes 88 CHAPTER 3 ANALYSING DATA e lst left primer ype primer_bind esidue 8 740 gt
15. account e g if you want to access an account from an additional com puter 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 Add New Account Create a new account on the server O This account already exists just connect Username Password Confirm password Email address optional C Connect every time I run Geneious Y More Options Figure 9 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 e 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 9 1 MANAGING YOUR ACCOUNTS 115 gt Add New Account Create a new account on the server O This account already exists just connect Username Password
16. alignment algorithm exist including the one used in the popular alignment software ClustalX 23 Multiple sequence alignment in Geneious Multiple sequence alignment in Geneious is done using progressive pairwise alignment The neighbor joining method of tree building is used to create the guide tree 3 5 SEOUENCE ALIGNMENTS 77 As progressive pairwise alignment proceeds via a series of pairwise alignments this function in Geneious has all the standard pairwise alignment options In addition Geneious also has the option of refining the multiple seguence alignment once it is done Refining an alignment in volves removing seguences from the alignment one at a time and then realigning the removed seguence to a profile of the remaining seguences The number of times each seguence 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 seguences aligned 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 seguence 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 t
17. am 0 7 di Document 280 J Help Panel Panel Three Kingdoms Table myt RNA synthe 1 30 q p Transcript varust varats of MAPK 2 7 D Rewrknon Enzymes 4 Zooming D Tree Oocumeets 16 eee eae SS is eno ys ose tor 6d oa FOSIOUES OF DOOM Out 10 View an ee Alignment View Text View Notes entire sequence and all its sa annotatoes Buttons toe v eust GB O tera El Reverse Complement GP Tramsime gt gt H D controling room are postioned Bouse ty bls vuo Consensus MIOIITCARGGG GAAGOAGA RIIGGGRACC You can also hold AR and turn O tblasin muse M Cene 2 Cenone Nucdeotide D Popset O Pore C roues sw processing Gi serveture Grag to select a region You can P Tascromy Grag vp and Gown 10 select and v Pram eds across muitpie in an the Eat p y button enters edt modo and 4 el e Cursor before residue O original residue 0 20001000 0000 a Figure 1 1 The main window in Geneious 1 2 USING GENEIOUS FOR THE FIRST TIME 9 1 2 1 The Sources Panel The Sources Panel contains the service Geneious offers for storing and retrieving data These in clude your local documents including sample documents Server Databases UniProt NCBI Pfam and Collaboration All these services will be described in detail later in the manual For more information see section 2 1 1 1 2 2 The Document Table The Document Table displays summaries of downloaded data such as DNA sequences protein sequences j
18. and joining contigs where necessary Reject alignments which do not meet overlap requirements 3 8 2 Assembly to a reference sequence 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 Assembly Choose the name of the sequence you wish to use as the reference in the Align to referencce option and click OK One contig will be produced at most and this will display the reference sequence at the top of the alignment view with all other sequences below it See section 3 8 4 for details on identifying differences or SNPs When aligning to reference the sequences are not aligned to each other in any way each of them is instead aligned to the reference sequence independently and the pairwise alignments are combined into a contig Assembly to a reference should therefore not be used if differences between reads are of interest If you just wish to use a reference sequence to help construction of the contig then you should select all sequences and the reference but choose None for Align to reference 3 8 CONTIG ASSEMBLY PRO ONLY 95 3 8 3 Trimming Trimming low guality ends of seguences is normally performed before assembling a contig This is because the noise introduced by low guality regions can produce incorrect assemblies Th
19. any of your local documents and any Notes that you add can be treated as user defined fields for use in sorting searching and filtering your documents Where can I add Notes You can add a note to any of your local documents including molecular seguences phyloge netic trees and journal articles You cannot add notes to search results from NCBI or EMBL etc until the documents are copied into one of your local folders The Notes View All documents have a Notes tab in the document viewer panel Click on the tab to display the Notes view which will show you all the notes that are attached to your selected document s To add a note to your document select the Add a Note button on the toolbar and then choose from the available note types Selecting a note type will create an empty note of that type To fill the note just start typing values into the fields Dotplot View Seguence View Notes EX Add a Note Save ld Source x Source Name Sample DNA Date Acguired Modified From original This field must conform to the following constraints O must begin with z constrained constrained string strindl Text Note N x And my poor fool is hanged No no no life Why should a dog a horse a rat have life Note And thou no breath at all Thou lt come no more Never never never never never Pray you undo this button Figure 2 10 The Notes View 46 CHAPTER2 RETRI
20. 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 4 Bootstrapping is the statistical method of resampling with replacement To apply bootstrapping 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 obtained 4 Jackknifing is a statistical method of numerical resampling based on deleting a portion of the original observations for each pseudo replicate A 50 jackknife randomly deletes half of the columns from the alignment to create each pseudo replicate 3 6 6 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 suppo
21. can be rooted by adding an outgroup a species that is distantly related to all the taxonomic units in the tree A common format for representing phylogenetic trees is the Newick format 13 3 6 2 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 guickly be obtained by using this method without assuming a molecular clock 20 3 6 BUILDING PHYLOGENETIC TREES 81 3 6 3 UPGMA This clustering method is based on the assumption of a molecular clock 15 It is appropriate only for a guick 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 3 6 4 Distance models or molecular evolution models for DNA seguences The evolutionary distance between two DNA seguences can be determined under the assump tion of a particular model of nucleotide substitution The parameters of the substitution model define a rate matrix that can be used to calculate the probability of evolving 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 eguilibrium freguencies and relative substitution
22. 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 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 nodes you can further restrict your search with the second drop down box e is greater than gt e is less than lt e is greater than or egual to gt e is less than or egual to lt Likewise if you are searching on the Creation Date search field you have the following op tions e isbefore or on e isafter 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 32 CHAPTER 2 RETRIEVING AND STORING DATA various types of documents that can be stored in Geneious such as 3D Structure Nucleotide sequence and PDF see Figure 2 5 Match all E of the following Document type a is 3 3D structure M Include Subfolders A Fewer Options 3 Name Summary Tm af Alpha Helix Alpha Helix Cyclopropane Cyclopropane af VirusVIRAL PROTEIN VirusVIRAL PROTEIN 1G5G ci lt gt
23. can read more about the e value in subsection 2 4 4 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 40 CHAPTER 2 RETRIEVING AND STORING DATA Match all of the following Any Field zs contains ES Document type geneious document type E Value First Author The article principal author uc CID The genbank ID of the sequence Height Tree Height Hit range Position of hit in result sequence Journal Title Where article was published Last Author The article last author Medline Date The article publication date Molecule Type The molecule type of the sequence Name name of a document No nodes Number of Nodes No tips Number of tips Organism Organism The organism of the sequence PDB name Name in PDB PMID The PubMed ID of the article Publication Date The article publication date Sequence Annotations sequence annotation text Sequence Length Residue length of the sequence Sequence Residues Size Size of the document Summary summary of a document a URL A url link to the published article v LU eSelect a document from the pane Figure 2 7 Searching the local documents on a user defined field 2 5 3 Checking and changing the location of your Local folders To check where your Local folders are bei
24. characters otherwise the gap character is treated like a normal residue 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 seguences contain guality information in the form of chromatograms you can select Highest Ouality to calculate a majority consensus that takes the relative residue guality into account When Highlight disagreements is checked the residues in the alignment that are identical to the consensus state for that column are grayed out This allows you to guickly locate variable sites in the alignment Similarly Highlight agreements greys out residues that are not indentical to the consensus allow ing you to guickly locate conserved sites in the alignments Highlight transitions transversions greys out residues that are not transitions transversions com pared to the consensus seguence When highlighting transitions transversions it is recom mended you turn on the ignore gaps consensus option or some residues may be wrongly high lighted due the consensus displaying N for sites that contain gaps and non gaps Highlight ambiguities greys out non ambiguous residues Go to next disagreement agreement transition transversion ambiguity goes to the next highlighted feature as described in the previous section on highlighting Zoom options These are a few options that can be turned on or off Auto zoom t
25. 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 10 and PAM 2 matrices Note The BLOSUM matrix is a substitution matrix The number of a BLOSUM matrix indicates the threshold similarity between the sequences originally used to create the matrix BLO SUM matrices with higher numbers are more suitable for aligning closely related sequences 3 5 SEOUENCE ALIGNMENTS 73 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 seguences This is done by inserting gaps in order to maximize the alignment score If the seguences are related along their entire seguence a global alignment is appropriate However if the relatedness of the seguences 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 16 and their algorithms was later extended by Gotoh to model gaps more accurately 6 For local alignments the Smith Waterman algorithm 21 is the most commonly used See the references provided for further information on these
26. 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 server database belongs to this group with a role of Edit unless you enter them into the g user 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 SQL 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
27. for the backbone Visibility e Filter atoms lets you set up rules to specify which parts of the molecule to render The following types of rules are available Atom number lets you show atoms based on their index in the structure document Chain lets you show atoms based on what chain they are in 3 1 DOCUMENT VIEWERS IN GENEIOUS 65 Secondary structure lets you show atoms based on what type of secondary structure they are a part of Temperature B factor lets you show atoms based on their Debye Waller factor which is usually measured in an X Ray crystallography to infer the 3D structure of the molecule Element lets you select specific atomic elements Hydrogen Oxygen etc Amino acid type lets you show atoms whose containing amino acid has specific prop erties Amino acid lets you show atoms belonging to specific amino acids Nucleotide lets you show atoms belonging to a specific nucleotide or nucleotide type Group type lets you show atoms that are part of a specific chemical group such as an amino group e Show all shows all atoms again 3 1 4 Tree viewer The tree viewer provides a graphical view of a phylogenetic tree Figure 3 6 When viewing a tree a number of other view tabs may be available depending on the information at hand The Sequence View tab will be visible if the tree was built from a sequence alignment using Geneious The Text View shows the tree in te
28. for the phylogenetic analysis of protein coding seguen VI Choosing appropriate susi een Shayo Andrew Rambaut amp Alexei Diummond 2095 Mol Bol Evol 2307 9 0 N Tree measures and the number of segregating sites in time structured population samples oa OE eee A Roald Forsberg Alexei J Drummond amp Jotun Hein 2004 BMC Genet 6 35 Molecular phylogeny of coleoid cephalopods Mollusca Cephalopoda using a multigene approach x effect of data partitioning on resolving phylogenies in a Bayesian framework A Molecular phylogeny of coleoi Figure 2 6 Advanced Search 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 and Pfam databases You can access these databases through the web at http www ncbi nlm nih gov http www uniprot org and http www sanger ac uk Software Pfam respectively These are all well known and widely used storehouses of molecular biology data When viewing data from a public database such as NCBI the data can not be modified This is demonstrated by the small padlock icon which appears in the status bar When this icon is present items cannot be added or removed from the table and they cannot be modified in any way To modify an item you must first move it to your local folders 2 4 1 Pfam See chapter 6 2 4 2 UniProt This database is a comprehensive catalogue of protein data It includes protein se
29. frame plugin PDF importer PHYLIP exporter PileUp importer RSF importer Seguence Alignment Seguence Concatenation Text Viewer Tree builder Description Provides importing and exporting support for a variety of 3 D structure fil Imports Chromatogram documents Provides Clustal W import Builds alignments using ClustalW Allows you to share your documents with other geneious users and to acce Provides DNA Strider import A viewer which displays the entire source xml for documents This is not i Provides a graphical view of a pair of sequences Provides access to EMBL s database at http www ebi ac uk embl Provides import and export functions for Endnote libraries Provides FASTA file importing exporting facilities Generates a single sequence representing the most common residues at e Provides a graphical view of seguence documents Provides a graphical view of a tree s document A plugin for viewing 3D proteins structures Select a 3D structure documen Provides MEGA export Provides access to NCBI s database at http www ncbi nih gov New seguence documents can be made by pasting or typing residues Provides Newick importing facilities Provides Nexus importing exporting facilities ORF analysis tools Imports PDF documents Provides Phylip export Provides PileUp import Provides RSF import Provides pairwise and multiple seguence alignment algorithms Provides the ability to concatenate seguences or seguen
30. genome Ed NC_001722 Human immunodeficiency virus 2 complete genome Ed NC_001664 Human herpesvirus 6 complete genome A Figure 2 4 The Search tab of the Document Table 2 3 SEARCHING 31 2 3 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 This feature provides more search options to select from Geneious allows you to search with a range of criteria however these depend on the database being searched All the fields in the NCBI public databases can be searched in any combination Each database has a specific list of fields and it is important to familiarize yourself with these fields to make full use of the Advanced Search The fields available for a search can be found in the left most drop down box after enabling the advanced search options Note When searching the Genome database 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 There are also Download items in the File menu and in the popup menu when document summary is right clicked Ctrl Click on MacOS The size of these files is not displayed in the Documents Table Be aware that whole genomes
31. ht tp www gnu org copyleft gpl html Install and start the server on one computer and then enter that com puter 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 Jabber server except possibly under a contracting agreement Chapter 10 Cloning Pro only Restriction Enzymes cut a nucleotide seguence 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 TTC Cit The cloning features in Geneious allow you to identify candidate Restriction Enzymes for your experiments and to determine in silico where they would cut your nucleotide sequences and which fragments they would produce It also lets you ligate fragments and insert a fragment into a vector If you select a nucleotide sequence restriction analysis is available under the menu item Tools Restriction Analysis and in the context menu right click on the sequence or Ctrl Click on MacOS e Find Restriction Sites allows you to specify an arbitrary candidate set of restriction en zymes and the desired number of matches so that you can e g identify enzymes that cut onl
32. in Geneious 3 1 1 The Sequence and alignment Viewer The Sequence view tab in the Document Viewer panel is available for Nucleotide sequences Protein sequences Alignments and some 3D structure documents If an alignment is selected this will be called Alignment View or Contig View if a contig is selected The options avail able are grouped under headings Zoom level Annotations Colors Layout Zoom options and Statistics The presence of these options varies with the kind of sequence data being viewed Zoom level The plus and minus buttons increase and decrease the magnification of the sequence by 50 or by 30 if the magnification is already above 50 P zooms to 100 The 100 zoom level allows for comfortable reading of the seguence 53 CHAPTER3 ANALYSING DATA oo Extract E Reverse Complement amp Translate P Edit b J Annotation Save i 33 66 99 i32 i65 igs 231 264 29 i i i i i i a W Zoom Level Ee A 1 Cys peroxiredox E emis C Compress annotations 330 363 396 429 462 495 528 561 594 27 mmm 11151 DEMI S a osiin mus JONIN KIEN Von kh source M mRNA 1 Gee _LL LI o C 1 Cys peroxiredoxin protein exon 3 4 source 1 MIEN 660 693 726 59 92 825 858 En 2 5 I Show AN Hide Alt exon 3 gt i E v M Colors 390 1 023 1 056 1 089 1 122 1 155 1 188 1 221 1 254 ot ee TE K UU TN i N E TIIA N
33. n db Create Note Type Delete Note Type Figure 2 12 The Edit Note Types window 2 8 NOTES 47 Creating Note Types Geneious does not restrict you to the note types that it comes with You can create your own note types to store any information you want To create a new note type click on the Create Note Type button in the left hand panel of the Edit Note Types window This creates a new note type with one empty field and displays it in the panel to the right Note The Note Type Name and Note Type Description fields distinguish your Note type from other user defined note types They do not have any constraints Here are some examples of Note Types Name Description Protein size Size of the protein in kDa Tree building method Method used to build tree UPGMA Neighbor joining Next you need to decide what values your Note Type will store by specifying its fields 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 Note Type to add or remove a field from the note 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 2 12 Constraints These are limiting facto
34. on a seguence position or annotation or select a region to zoom in Alt right click to zoom out Figure 3 2 The similarity graph for an alignment of nucleotide seguences 3 1 DOCUMENT VIEWERS IN GENEIOUS 57 Seguence Logo This is available for seguence alignments It displays a seguence logo where the height of the logo at each site is egual 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 he horizontal width of each site is less than one pixel then the height is the average of the information 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 Hydrophobicity This is available with protein seguences It displays the Hydrophobicity of the residue at every position or the average Hydrophobicity when there are multiple seguences pl pl 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 Sliding window size 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 o
35. or USB drive so you can access it from other computers To store your data on a different drive simply click the Select button in the welcome window and choose an empty folder on your 8 CHAPTER 1 GETTING STARTED drive where you would like to store your data The data location can also be changed later by going to the General tab under Tools Preferences in the menu and changing the Data Storage Location option Geneious will offer to copy your existing data across to the new location if appropriate 1 1 2 Upgrading to new versions To upgrade existing Geneious installations simply download and install the new to the same location This will retain all your data 1 2 Using Geneious for the first time Figure 1 1 shows the main Geneious window This has six important areas or panels pa ee ei Bak forward Seah Agents Algemene Tree Assembly Primers Cloning Help 1969 seleci s Me v D tocal 00 Name Duscripaon how Sia 1 Helo v D Sample Documenti 00 Aignan of Arma Se Algrmart of 10 sequences Chicken Fro 708 D 30 Sarvetures 7 COKE CDS Muttipie alignment of 51 Cytochrome 0 705 O Agamer 9 env abgrenert Migrmert of sequences AB234101 1 479 Alignment View Help D Contig Assembly Massing arnotaton Alignment of 2 sequences houie mouse 2 44 eeBee eee Poratie protein peptidase from kiw 221 me e uno ma People sequences from f 426 4 Sources m
36. part the differences between fragments is This can also be done against a 3 8 CONTIG ASSEMBLY PRO ONLY 93 known reference seguence when differences between each of the fragments and the reference are of interest 3 8 1 Assembling a Contig To assemble a contig firstly select all of the seguences you wish to assemble in the document table and click Assembly in the toolbar in the Tools menu or in the popup menu right click ctrl click on Mac OS on the documents The basic options for contig assembly will then be displayed g Assembly Minimum Overlap Overlap Identity Assemble to reference None Trim Sequences Maximum Ambiguous Bases Screen for vectors Univec High sensitivity Finds regions which are likely to be vector contamination using an implementa O Do not trim Y More Options Figure 3 17 Basic assembly options The options available here are as follows e Minimum Overlap The minimum overlap in nucleotides between a seguence and any seguence in the contig reguired for the seguence to be included in the contig e Overlap Identity The minimum identity in percent of the overlap region between a seguence and any seguence in the contig reguired for the seguence to be included in the contig e Align to reference Select a seguence to use as the reference See section 3 8 2 e Trim Seguences Select how to trim the ends of the seguences being assembled See section 3 8
37. rates Eguilibrium freguencies 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 74 nc TG mr that sum to 1 Relative substitution rates define the rate at which each of the transitions A G C T and transversions A C A e T C G G gt T occur in an evolving sequence It is represented as a 4x4 matrix with rates for substitutions from every base to every other base Jukes Cantor This is the simplest substitution model 11 It assumes that all bases have the same eguilibrium base freguency i e each nucleotide base occurs with a freguency of 25 in DNA seguences and each amino acid occurs with a freguency of 5 in protein seguences This model also assumes that all nucleotide substitutions occur at egual rates and all amino acid replacements occur at egual rates HKY The HKY model 9 assumes every base has a different eguilibrium base freguency and also assumes that transitions evolve at a different rate to the transversions Tamura Nei This model also assumes different eguilibrium base freguencies In addition to distinguishing between transitions and transversions it also allows the two types of transitions A G and 82 CHAPTER3 ANALYSING DATA CT to have different rates 22 3 6 5 Resampling Bootstrapping and jackknifing Resampling is a statistical technique where a procedure such as phylogenetic tree
38. same if you are searching your local documents or a public database such as NCBI To search the selected database or folder click the Search button from the toolbar For non local folders search will be on by default and cannot be closed This applies to NCBI and EMBL databases For local folders search is off by default When search is first activated the document table will be emptied to indicate no results have been found To return to browsing click the Search button again or press the Escape key while the cursor is in the search text field To initiate a search enter the desired search term s in the text field and press enter or click the adjacent Search button Once a search starts the results will appear in the document table as they are found 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 presented in the status bar directly below the toolbar immunodeficiency N N J C Search More Options Name Summary R E NC 001802 Human immunodeficiency virus 1 complete genome E NC 004455 Simian immunodeficiency virus 2 complete genome Ed NC_001549 Simian immunodeficiency virus complete genome Ed NC_003074 Arabidopsis thaliana chromosome 3 complete sequence E NC 002305 Salmonella typhi plasmid R27 complete seguence a NC_001870 Simian Human immunodeficiency virus complete
39. selected This will display the alignment in the sequence viewer If you are using Geneious pro there is also an option to perform the alignment using ClustalW an independent and widely used alignment program Figure 1 3 The Help Panel 1 2 5 The Toolbar The toolbar gives quick access to commonly used features in Geneious including the Search for documents by keywords Agents that search databases for new content even while you sleep Sorting sequences by similarity pairwise or multiple sequence Alignment Tree building and Help For more information on the toolbar see section 2 1 5 1 3 TROUBLESHOOTING Geneious pro trial 1 3 5 a o gt PB UT 7 amp Back Forward Search Agents Note Sort Alignment Tree Completed found 23 documents Sources v Local 32 p 51 185 22 DefaultPhylogenyDocumentTest 14 p 58 v HIV 0 nef 40 pg 79 v 2 NewcastleDisease 1 lentogenic1 23 lentogenic2 19 v sample Documents 0 O 3D Structures 2 Alignments 82 O Nucleotide Documents 4 Protein Documents 2 5 Tree Documents 2 PR iiaa JON cf 7229463 Figure 1 4 The Toolbar 1 2 6 The Menu Bar The Menu Bar has seven main menus File Edit View Tools Sequence Collabora v Back v History v Forward v Search v Agents v Divider 1 v Note v Sort v Divider 2 v Alignment v Tree v Divider 3 v Help tion Help
40. sequence before opening the primer dialog then this region will automatically be used for Included Region and Target Region All of these are expressed in base pairs from the beginning of the sequence and are as follows 3 7 PCR PRIMERS PRO ONLY 87 e 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 seguence you wish to amplify and unless the advanced options allow otherwise the left and reverse primers must fall somewhere 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 left 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 Output from Primer Design
41. sure you are still connected to the Internet Both Dial up and Broadband can disconnect If you are connected then the error message indicates you are behind a proxy server and Geneious has been unable to detect you proxy settings automatically You can fix this problem 1 Check the browser you are using These instructions are for Explorer Safari and Firefox Q N Open your default browser Use the steps in Figure 1 5 for each browser to find the connection settings Now go into Geneious and select Preferences There are two ways to do this Shortcut keys Ctrl Shift P Windows Linux Command Shift P Mac OS X Tools Menu Preferences 5 This opens the Preferences Click on the General tab There are five options in the drop down options under Connection settings Figure 1 6 Use direct connection Use this setting when no proxy settings are reguired Use browser connection settings This allows Geneious to automatically import the proxy settings This may not work with all web browsers 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 Use SOCKS proxy server Autodetect Type 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 a SOCKS pro
42. te K ACC A gt M Zoom Options 1 Cys peroxiredoxin protein CDS Statistics 147 1596 Cys peroxiredoxin protein mR gt I Cys peroxiredoxin protein MRNA eng 147 E N N A irequencies prk Sl TEA ra ai te ed rire pal s Se ee a ta AA A 40 27 2 C 35 23 8 AAA G 38 25 9 T 34 23 1 GC 73 49 7 Selected 147 residues from residue 525 to 671 Figure 3 1 A view of an annotated nucleotide sequence in Geneious 3 1 DOCUMENT VIEWERS IN GENEIOUS 55 ma wu zooms out so as to fit the entire sequence in the available viewing area Zooming can also be guickly achieved by holding down the zoom modifier key which is the Ctrl key on Windows Linux or the option key on Mac OS and clicking When the zoom key is pressed a magnifying glass mouse cursor will be displayed e Hold the zoom key and left click on the seguence 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 and out e Hold the zoom key and click on an annotation to zoom to that annotation Colors The colors option controls the coloring of the seguence nucleotides or amino acids Uncheck the color checkbox to turn off all coloring without viewing further options Coloring schemes differ depending on the type of sequence For example the Polarity and Hydrophobicity coloring schemes are available only for Protein seguences Layout L
43. with this chapter as there are a number of formats to be aware of 2 2 IMPORTING AND EXPORTING DATA 23 2 2 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 format detection fails select the file type you wish to import Figure 2 3 The different file types are described in detail in the next section File name Import Files of type Cancel ace PHRAP File format ace ace 1 txt Chromatogram ab abi ab1 scf Clustal aln DNA Strider str DNAStar seg pro Endnote 8 0 or 9 0 xml Fasta Autodetect fasta fas fa mpfa fna fsa txt Figure 2 3 File import options 2 2 2 Datainput formats Geneious version 4 0 2 can import the following file formats CLUSTAL format The Clustal format is used by ClustalW 24 and ClustalX 23 two well known multiple se guence alignment programs Clustal format files are used to store multiple seguence alignments and contain the word clustal at the beginning An example Clustal file CLUSTAL W 1 74 multiple sequence alignment seql KSKERYKDENGGNYFOLREDWWDANRETVWKAITCNA seg2 YEGLTTANGXKEYYODKNGGNF FKLREDWWTANRETVWKAITCGA se
44. y 2 Retrieve single sequence per hit with COGs info O Retrieve entire cog per hit with COGs info Just display COGs info Figure 5 1 Configuring a COGs BLAST Chapter 6 Pfam Pro only Pfam is a large collection of multiple seguence alignments and hidden Markov models covering many common protein domains and families The data for Pfam is taken from seguences in UniProt Pfam can be found online at the following locations e Sanger Institute UK e Washington D C USA e Karolinska Institutet Sweden e Institut National de la Recherche Agronomique France 6 1 Setting up the Pfam databases At the time of release of Geneious 3 5 there was no public online interface to the Pfam database although there is one in the works at the Sanger Institute For this reason if you want to search the Pfam databases you will need to download them first As of Pfam 22 July 2007 the subset of the Pfam databases used by Geneious totalled about 4GB in size so it is recommended you download them somewhere with a fast connection You can use Geneious to search five of the Pfam databases 1 Pfam A seed 29 MB contains records on the manually curated domains in Pfam A and the seed alignment alignment of a representative subset of all occurrences of this domain in UniProt seguences for each domain 2 Pfam A full 392 MB contains records for the manually curated domains in Pfam A and the full alignment alignment of all occ
45. 132 CHAPTER 11 SERVER DATABASES PRO ONLY 11 4 2 Database Indexing Geneious indexes every document that is added to a server database for searching It is very unlikely that this index will become corrupted But if you are not getting correct search results or if you simply believe the database index has become corrupt somehow the admin of the Everybody group can right click on the top folder of a server database to re index it This will not affect any other users until it is complete however if your database contains many documents it will take a long time Geneious must be left open to re index the database Bibliography 1 SF Altschul W Gish W Miller EW Myers and DJ Lipman Basic local alignment search tool J Mol Biol 215 1990 no 3 403 410 16 18 25 35 2 MO Dayhoff ed Atlas of protein seguence and structure vol 5 National biomedical re search foundation Washington DC 1978 72 73 3 R Durbin S Eddy A Krogh and G Mitchison Biological seguence analysis Cambridge University Press 1998 76 4 J Felsenstein Confidence limits on phylogenies An approach using the bootstrap Evolution 39 1985 no 4 783 791 82 5 DF Feng and RF Doolittle Progressive sequence alignment as a prerequisite to correct phyloge netic trees J Mol Evol 25 1987 no 4 351 60 76 6 O Gotoh An improved algorithm for matching biological sequences J Mol Biol 162 1982 705 708 73 7 S Guindon and O
46. ATA Chapter 4 Custom BLAST Pro only Custom BLAST allows you to create your own custom database from either FASTA files or seguences in your local folders and BLAST against it 4 1 Setting Up The Custom BLAST plugin reguires access to NCBI BLAST binary files 4 1 1 Setting up the Custom BLAST files yourself If you want you can download or otherwise acguire the NCBI BLAST binary files outside of Geneious You can download them from here ftp ftp ncbi nih gov blast executables 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 select the Custom BLAST service Click the Change Database Location and browse to the location of the files 4 1 2 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 select the Custom BLAST Service Click the Let Geneious do it button Then click the Start button 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 99 100 CHAPTER 4 CUSTOM BLAST PRO ONLY 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
47. Advanced Search criteria Look at an example of a new Note type called Protein size which takes a text value for the protein in kDa kiloDaltons see Figure 2 7 Important You must use quotation marks if and blank spaces are part of your search criteria No guotation marks lead to unreliable results Wild card searches When you are looking for all matches to a partial word use the asterisk For example typing oxi would return matches such as oxidase oxidation oxido reductase and oxide This is useful for performing generic searches You can also place the asterisk in the middle of the word but not at the beginning This feature is available only for local documents 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 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
48. CBI 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 key on Mac OS 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 2 5 STORING DATA YOUR LOCAL DOCUMENTS 39 2 5 2 Searching your Local folders The Services Panel allows you to browse your Local folder hierarchy Next to each folder name in the hierarchy is the number of documents it contains 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 You can search the Local folder and sub folders the same way you search the public databases by clicking on the Search icon If you have defined a new type of note in Geneious and a Note has been added it will also be added to the
49. Create from the agents dialog Choose a folder for the agent or create a new one and make sure that the Make destination folder a smart folder checkbox is checked When a folder is turned into a smart folder it is given a subfolder called reject At first all the documents delivered by the agent will be put in this folder Drag the documents that you want to keep into the main folder and future documents delivered by the agent will be compared to the accepted and the reject documents and stored in one or other of the two folders appropriately Make sure that you leave documents in the reject folder as smart folders need negative examples to build an accurate comparison model Note that unread documents in the main folder will not be compared while all documents in the reject folder will be 109 110 CHAPTER 7 SMART FOLDERS PRO ONLY Chapter 8 Geneious Education Pro only This feature allows a teacher to create interactive tutorials and exercises for their students A tutorial consists of a number of HIML pages and Geneious documents The student edits the pages and documents to answer the tutorial guestions and then exports the tutorial to submit for marking 8 1 Creating a tutorial The backbone of Geneious Tutorials are the HTML documents Simply create your documents and place them together in a folder If you make a page called index html it will be treated as the main page Geneious will follow all hyp
50. E ek ENSIN A SAK Cad Be WLI COGS so poe sa EARS Asa kse asa vas ui Pfam Pro only 61 Setting tip thie Piamdatabas s ow ee eh e ee YSE Rok ATK ee o G rem Decent eee ee Ka ra EE AE ed A WSN s RY 63 Piam Operations oca 64008 RARAS AAA RR A ERG HE RS OS Smart Folders Pro only Geneious Education Pro only SL Ceatnga PONG lt o lt asci peyro AAA EKKS he aE Dees Be eee A ONAL sk ea Ce ne Cee RR ae ee oe eared MIKE NOS Collaboration Pro only 9 1 Managing Your AOU oa gcc ku RE REDE RE OR ee hee ERS 92 Mange Your Cones lt lt e s bee Panis SEKIN ee A ee 93 Sharing Docente 20 1302383045 s RR ERS AAA Rae SDS 9 4 Browsing Searching and Viewing Shared Documents 103 103 104 105 105 106 107 109 111 111 112 CONTENTS 10 11 or Chab ge KS i tanaan Kin hp owt Josey a ee het ee ear J min als oe wed Cloning Pro only 10 1 Find Restriction Sites 2 640406 06 6 44 a ea we ee es 102 Dicest imo ee oa RAE A RRA AA 10 3 Insert into Vector s mum ee n k kela a ee aiel sai W X Server Databases Pro only 11 1 Supported Dalobase Systeme sis kuissa Ta yu Paas SEE N 135 SIIS MP aurea EP HS EDS AR HP Aokin Ee Eee Oe ES x 11 3 Removing a server database gt o acoc ca k kee rasada ee eRe A 11 4 Adminstration 2 i dica dae ee Anas eB Oe RD ee EO owe e a 119 121 122 123 125 CONTENTS Chapter 1 Getting Started One of the best ways to get an introd
51. EVING AND STORING DATA 2 8 1 Editing Notes To edit the fields of a note simply click on the field and enter your data Some fields may have constraints which you can edit in the Edit Note Types dialog see 82 8 2 If the data you have entered does not conform to the constraints of the field it will be displayed in red and you will be shown the field s constraints in a tooltip see figure 2 10 Tip To enter a new line in a text field press shift enter or ctrl enter When multiple documents are selected the Notes view displays all of the notes belonging to the selected documents When each document has the same value for a note field it is displayed in the viewer If the documents have different values or some of the selected documents do not have a note of that particular type then the field will show that it represents multiple values Changes made to the fields will apply to all selected documents 2 8 2 Editing Note Types To edit your note type click the Add a Note button on the viewer toolbar and select Edit note types This will bring up a window similar to that displayed in figure 2 12 FX addanote M COGs Note Figure 2 11 Edit Note Types 2 Edit Note Types Existing Note Types Name Source Description Describes the source of a document constrained Source Name Date Acguired Modified from original dh Date Modified m Text True False Whole Number Decimal Date Drop down list
52. GATCGCG CGATATCATC SACTGCGCO ity of Auckland 1001 Auckland New Zesland uk arvan ign Taen ate Tomi D anerntieres 7 r a lt j n a see ANCSACICAC BACCECAMES ACCMACSAME CCCCACCAMC CAMCAMES sea We ieee ainar ate of is arpst an say o EN ose arez SEEN ern parta seer PMID 12136032 003 fa A Son Bose Evanch Selected 258 residues from residue 21 to 278 a Nucleotide sequence b Journal Article c Phylogenetic tree Figure 1 2 Three document viewers 1 1Tn 10 CHAPTER 1 GETTING STARTED 1 2 4 The Help Panel The Help Panel has two sections Tutorial and Help The tutorial gives you hands on experience with some of the most popular features of Geneious The Help section displays a short description of the currently selected service or document viewer This panel can be closed at any time by clicking the X symbol in its top corner or by toggling the Help button in the Toolbar If you are new to Geneious working through the tutorial is a great way to familiarize yourself with Geneious O Help Previous 70f21 Next Sequence alignment Geneious allows you to align a set of sequences If the sequences you just imported are not selected select them all now Click on the Alignment button in the toolbar Click OK to accept the default alignment settings When the alignment has finished a new alignment document will be added to the current folder and
53. Gascuel A simple fast and accurate algorithm to estimate large phylogenies by maximum likelihood Syst Biol 52 2003 no 5 696 704 80 8 M Vingron HA Schmidt K Strimmer and A von Haeseler Tree puzzle maximum likelihood phylogenetic analysis using guartets and parallel computing Bioinformatics 18 2002 no 3 502 504 27 9 M Hasegawa H Kishino and T Yano Dating of the human ape splitting by a molecular clock of mitochondrial dna J Mol Evol 22 1985 no 2 160 174 81 10 S Henikoff and JG Henikoff Amino acid substitution matrices from protein blocks Proc Natl Acad Sci USA 89 1992 no 22 10915 10919 72 73 11 T Jukes and C Cantor Evolution of protein molecules pp 21 32 Academic Press New York 1969 81 12 S Kumar K Tamura and M Nei Mega3 Integrated software for molecular evolutionary ge netics analysis and sequence alignment Brief Bioinform 5 2004 no 2 150 163 29 133 134 BIBLIOGRAPHY 13 DR Maddison DL Swofford and WP Maddison Nexus an extensible file format for sys tematic information Syst Biol 46 1997 no 4 590 621 27 29 80 14 JV Maizel and RP Lenk Enhanced graphic matrix analysis of nucleic acid and protein se quences Proc Natl Acad Sci US A 78 1981 no 12 7665 9 70 71 15 C Michener and R Sokal A quantitative approach to a problem in classification Evolution 11 1957 130 162 80 81 83 16 SB Needleman and CD Wunsch A gen
54. Geneious 4 0 2 Biomatters Ltd 17th September 2008 Contents 1 Getting Started 7 11 Downloading amp Installing Geneious lt lt lt cocos cir Rs Van ks 7 1 2 Using Geneious tor the first ime o se lt a ED EES PUNK Gn ES 8 L3 TODO os cee kka RARA OSS AA 11 2 Retrieving and Storing data 15 2l Thenon a AAA e t 15 22 importing and exporting data ece 2445 em k a hh opaa i KA PKA 22 29 Dearing os soo ee CAE ye RSE KE ER DAG Ew ERE e EERE BS 30 A so eo boa Se ote POH eS wee 2 ORE YS EASE Hae SEs 32 25 Storing date Your Local Documents lt lt 24 2944 85 jka ORE eS 37 El MENNE 3052 sua pi oe et BE eR SSE EES s Sa dd e KS 41 27 Pilterng and Similarity sorting aooi rocon Be ee eS we 44 2o DNOS IE 45 29 PEREDES oa ged Oe ee ee Re ee ee p a ea PRN ee ee O ek 49 210 Tenn and Saving Nee ar a X 51 3 Analysing Data 53 3 1 Document Viewers in Geneious o e knn 53 J2 Liki ci cok eh ee tans 32 08 26 ee e a sah Doha osak Pot 8 69 J SIIRI in ie e La oe Ee SIEN Chee eed Pe x N 70 CONTENTS 34A DOO II D TOAS AIRI lt a egona d vaa PKR Oe ee KUKA E eS 3 6 Building Phylogenetic tees o cs 0 ce ER KK KM KSK RW He ER Re EO Oy PORPOMES UT 0344032 EES SHEE HA JA ERE ES ao Contig Assembly Pro only acid aa s Es Ws POS kakka 8 ge TSS AIN ss Kanaa MA E REEN SSE Kasta ae oN wo Custom BLAST Pro only TI SINI sa ksi gaa e ES YEE EER BET EM k lkk Ws A E COGs BLAST Pro only SL TIE Pk Kon oe eee Bele A k KO
55. T database setup dialog will come up automatically when a COGs BLAST is attempted and the COGs database is not set up Select any seguence in the document table right click it and select COGs Blast Click the Let Geneious do it button Then click the Start button After a few seconds the COGs BLAST database files will start downloading You can click Pause to pause the download Once all the files have finished downloading aned settung up you will need to close the dialog If you shut down Geneious with a file partially downloaded you will need to start downloading it again from the beginning Files completely downloaded will not need to be downloaded again 5 2 BLASTing COGs Select any seguence in the document table right click it and select COGs Blast Geneious will give you several options for your blast see Figure 5 1 Number of hits to fetch allows you to fetch results for the best n hits for your sequence You can choose to download COGs sequence from NCBI with full annotations or to load them without annotations from the COGs database file Finally you have the option of retrieving the sequences for your hits the entire COG for each hit or to just display information about the hits Once you have made your choices click OK If you have selected a Nucleotide sequence Geneious will give you options to translate it at this point 270065 Blast Number of hits to fetch 18 Retrieve sequences from NCBI
56. TER 2 RETRIEVING AND STORING DATA e Go To Next Disagreement see section 3 1 1 e Preferences see section 2 9 2 1 8 Sequence Menu This contains several operations that can be performed on Protein and Nucleotide sequences as Well as Sequence Alignments in some cases e New Sequence create a new nucleotide or protein sequence from residues that you can paste or type in e Extract Region Reverse Complement Translate see section 3 1 1 for details Sometimes a selection in the sequence viewer is required before performing these e Find ORFs Finds all open reading frames in a sequence and annotates them e Trim Ends See section 3 8 3 e Change Residue Numbering changes the original residue numbering of the selected sequence e Convert between DNA and RNA changes all T s in a sequence to U s or vice versa de pending on the type of the selected sequence Once this is performed click Save in the Sequence View to make the change permanent Collaboration Menu Pro only This contains actions that can be performed with Collaboration accounts which allow you to share you work with other Geneious users Help Menu This consists of the standard Help options offered by Geneious 2 2 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
57. ad e Table Columns contains the same functionality as the popup menu for the document table header See section 2 1 2 for more details e Viewers contains a list of available document viewers Clicking one will select the view if it is available on the currently selected documents e Open document in new window Opens a new window with a view of the currently selected document s 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 2 1 THEMAIN WINDOW 21 e Document Windows Lists the currently open document windows Selecting one from this menu will bring that document window to the front Tools Menu e Alignment see section 3 5 e Tree see section 3 6 e Assembly see section 3 8 e Primers see section 3 7 e Cloning see section 10 e NCBI Blast Perform an NCBI Blast search using the currently selected sequence as the query See section 2 4 4 e Pfam see section 6 e COGs Blast see section 5 e Linnaeus Blast Perform a blast search and display the results using the Linnaeus viewer Evolutionary trees are built for hits within the same species These are then displayed inside boxes nested according to the NCBI taxonomy e Create BLAST Database see section 4 1 3 e Extract Annotations Search the selected sequences or al
58. ade by the base calling software Layout Options controlling layout and view Those include X and Y axis scaling size of largest base letter when Seguence logo is on and minimum size of base letter to prevent bases of low guality becoming unreadable 3 1 6 ThePDF document viewer To viewa pdf document either double click on the 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 3 1 7 The Journal Article Viewer This viewer provides two tabs Text View and BibTex Text view displays the journal article details including the abstract The text contains a link to the original article through Google Scholar below the title and authors Figure 3 8 BibTex is the standard ISTFX bibliog raphy reference and publication management data format ATEX is a common program used to create formatted documents including this one The information in the BibTex screen can be exported for use in TEX documents 3 2 Literature 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
59. algorithms Pairwise alignment in Geneious A dotplot is a comparison of two seguences A pairwise alignment is another such comparison with the aim of identifying which regions of two seguences are related by common ancestry and which regions of the seguences have been subjected to insertions deletions and substitu tions The options available for the alignment cost matrix will depend on the kind of seguence e Protein seguences have a choice of PAM 2 and BLOSUM 10 matrices e Nucleotide seguences 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 gt 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 e 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 The score of a pairwise alignment is matchCount matchCost mismatchCount mismatchCost For each gap of length n a score of gapOpenPenalty n 1 gapExtensionPenalty is subtracted from this Where 74 CHAPTER3 ANALYSING DATA e gapOpenPenalty Th
60. and the name of the Journal When a document is selected the abstract of the article is displayed in the Document Viewer 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 Note If the full text of the article is available 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 As well as the abstract and links Geneious also shows the summary of the journal article in 70 CHAPTER 3 ANALYSING DATA A virus reveals population structure and recent demographic history of its carnivore host Roman Biek Alexei J Drummond amp Mary Poss Wildlife Biology Program University of Montana Missoula MT 59812 USA rbiek emory edu Science 2006 311 538 41 Google Scholar Directly transmitted parasites often provide substantial information about the temporal and spatial characteristics of host to host contact Here we demonstrate that a fast evolving virus feline immunodeficiency virus FIV can reveal details of the contemporary population structure and recent demographic history of its natural wildlife host Puma concolor that were not apparent from host genetic data and would be impossible to obtain by other means We suggest that rapidly evolving pathogens may provide a complementary tool for studying population dynamics of their hosts in
61. ations valid for your current selection 86 CHAPTER3 ANALYSING DATA 3 7 1 Design Primers The Primer Design dialog which is then displayed contains two main areas Design Primers Select Task s Design New s Design with Existing Design a pair of primers Design primer pair with probe Region Input Options e C Target Region Product Size Between 100 And 3004 C Optimal Product Size Number of Pairs to Generate se Y More Options Figure 3 13 The primer design dialog Task Two tasks are available 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 design 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 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 36bp in length or less Region Input Options 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
62. ayout has various options controlling the layout of the seguence e Show tree This toggles the display of the phylogenetic tree when viewing the alignment of a phylogeny document e Show residue positions This toggles the display of the residue position number above the seguence residues e Show original seguence positions This toggles the display of the residue position numbers for the original seguence on a per seguence basis It is only available for alignment docu ments and seguences that were extracted from other seguences e Show space every 10 residues If you are zoomed in far enough to be able to see individual residues then an extra white space can be seen every 10 residues when this option is selected e Wrap sequence This wraps the sequences in the viewing area A shortcut is to click the layout check box without expanding it 56 CHAPTER 3 ANALYSING DATA e Wrap on 10 residue boundaries This is automatically turned on if the wrap sequence option is on and will force the sequence wrapping to occur in multiples of 10 nucleotides or amino acids e Show sequence and graph names Show or hide sequence and graph names inside the se quence viewer panel Graphs This option is visible when viewing protein sequences chromatogram traces multiple se quences or sequence alignments Turn this option on by clicking the Graph checkbox and the graph s will be displayed below the sequence s The number to the right of eac
63. ble actions are Extract Extract the selected part of a sequence or alignment into a new document Reverse Complement Reverse sequence direction and replace each base by its complement This is available only for nucleotide sequences Translate Translate DNA into protein Clicking on this choice brings up a list of genetic codes that can be used Choose the appropriate one and click OK This is available only for nucleotide sequences Edit Annotations and Save 3 1 DOCUMENT VIEWERS IN GENEIOUS 61 Editing sequences and alignments To edit sequence s or an alignment click the Edit toolbar button After selecting a residue or a region you can either type in the new contents or use any of the standard editing operation such as Copy Ctrl C Cut Ctrl X Paste Ctrl V and Undo Ctrl Z All operations are under the main Edit menu Selecting a region enables the Annotations button as well which opens an annotation en try dialog Enter an annotation name and select a existing type or type a new one Click on More Options to enter additional properties for that annotation Double click on an existing annotation to edit it or right click Ctrl click on MacOS to display a pop up menu to delete annotations You can also copy an annotation from one sequence to another from the pop up menu When editing an alignment it is possible to select a region which may span several sequences and drag it to the left or right Dra
64. ce alignments A viewer which shows a simply text view of any document type Provides methods for building trees from alignments fox gt Cancel Apply Restore defaults Author s Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Andrew Rambaut Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Biomatters Figure 2 14 The plugins preferences in Geneious Version F 1 3 5 1 3 5 3 3 5 1 3 5 0 9 2 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 0 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 PA 2 10 PRINTING AND SAVING IMAGES 51 Appearance options allow you to change the way the main toolbar and the document table look Behaviour options allow you to change the way newly created documents are handled Such as whether they are selected straight away and where they should be saved to 2 10 Printing and Saving Images Geneious allows you to print or save as an image the current display for any document viewer This includes the seguence viewer tree view dotplot and text view 2 10 1 Printing Choose print from the file menu The following options are available Portrait or landscape Controls the orientation of the page
65. choose a digested fragment or a seguence with two restriction site annotations to use as an insert and insert them into a vector circular seguence 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 The following sections explain the more complicated operations in a little more detail 10 1 Find Restriction Sites 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 seguence You can configure the following options Figure 10 1 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 will always include the option to use all known commercially available restriction enzymes but if your search index is intact then all restriction enzyme set documents from your local database will also be listed see below for how to create such a document e Minimum effective recognition seguence length lets you filter the candidate enzymes to in clude only ones whose recognition seguence has a given minimum effective length For example EcoRI s recognition seguence is 6 nucleotides long GAATTC The effective length takes ambiguities into account so that e g the seguence YS only has an effective length of 1 it is a better measure for the expected number of hits in a random se
66. complete All of the same options available for designing primers also apply to testing so if the primers are expected to bind to guite different regions of the test seguences the primer binding region may have to be extended and the target region option can be omitted Click the OK button and testing will commence Once complete a dialog will present the results This dialog tells you how many of the seguences were compatible with the specified primers and probes and provides details and choices very similar to the one described in sec tion 3 7 1 The compatible primers can be annotated onto the seguences in a similar manner to that when designing primers Additionally if the primer seguences were not already annotated with a primer annotation they will be annotated during testing 90 CHAPTER3 ANALYSING DATA 3 7 3 Primer Characteristics Geneious can determine the primer characteristics of sequences such as melting point To do this select any number of sequences that are 36 base pairs or fewer in length and choose the same Primers action as you do with design or test then choose Primer Characteristics from the popup menu that appears If you select just two seguences you have the additional option of determining their pair characteristics Determining the pair characteristics of two primer seguences can be used to see if two seguences can pair and how well they do so 3 7 4 Advanced Options The parameters which are use
67. 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 Currently you can use Geneious to read store and open PDF files and future versions will have more options for storing and manipulating PDF Phrap Ace files 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 28 CHAPTER 2 RETRIEVING AND STORING DATA PileUp format The PileUp format is used by the pileup program a part of the Genetics Computer Group GCG Wisconsin Package PIR NBRE format Format used by the Protein Information Resource a database established by the National Biomedical Research Foundation Raw sequence format A file containing only a sequence Rich Sequence format RSF Rich Sequence Format files contain one or more sequences that may or may not be related In addition to the sequence data each sequence can be annotated with descriptive sequence information Sequence 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 again
68. d to pick primers and DNA probes are highly customisable through the advanced options section of the primers dialog To access this select part of a seguence for testing or designing and select Primers from the menu as detailed above Now click the More Options button and a the advanced options will appear below the standard options Additional Options The advanced options include additional options that tell Geneious to be more lenient with how it designs and tests primers e Maximum Degeneracy Turning this on allows Geneious to design primers which con tain a certain number of ambiguities Such a primer is called a degenerate primer This is because the seguence actually represents more than one primer seguence The maxi mum degeneracy that you specify is the maximum number of primers that any primer seguence 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 2 8 because R represents the two bases A and G e Maximum Mismatches This is available when testing and allows you to specify a limited number of mismatches that you wish to permit between a primer and the target seguence You can limit the position in which mismatches are allowed by clicking the Mismatch Options button e Inverse PCR Enables inverse PCR which will inver
69. direc tions Green is used for regions with reads in both directions and yellow is used for regions with reads in one direction only Consensus Pro only This option is available when viewing alignments When checked the viewer displays the consensus sequence with the aligned sequences The consensus sequences has the same 58 CHAPTER3 ANALYSING DATA length and shows which residues are conserved are always the same and which residues are variable A consensus is constructed from the most freguent residues at each site align ment 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 fractional support for each nucleotide in the ambiguity set A and G in this case thus e g two rows with R are counted the same as one row with A and one row with G When more than one nucleotide is necessary to reach the desired threshold this is represented by the best fit ambiguity symbol in the consensus for protein seguences this will always be an X In the case of ties either all or none of the involved residues will be selected Hence an alignment column with only As and Gs in egual number will be represented as an R in the consensus seguence regardless of the consensus threshold When ignore gaps is checked the consensus is calculated as if each alignment column consisted only of the non gap
70. download the databases elsewhere and then transfer them to your computer You may also consider down loading all databases except Pfam A full 6 2 Pfam Document Types There are three special document types used for Pfam data He S Pfam seguence documents are based on UniProt seguences They contain all the informa tion from the UniProt seguence plus information on the Pfam domains in the seguence You can view the domains as annotations in the seguence view or on their own from the domain View Domain documents contain information about Pfam A full Pfam A seed and Pfam B do mains This includes general information about the domain references visible in the reference view and the alignment for the domain gt Clan documents contain information about a clan including general information refer 6 3 PFAM OPERATIONS 107 ences visible in the reference view and a list of the domains which are members of this clan 6 3 Pfam Operations There are a number of special operations available to Pfam documents and UniProt seguences To take advantage of these operations you will need to have the Pfam databases set up The following Pfam operations are available e Create Pfam Seguence creates a Pfam seguence document from a UniProt seguence You can view the domain information in a Pfam seguence document using the Domain Viewer This operation can take a long time e With Find Similar Seguences you can search and create docum
71. e 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 usefulif you are aligning seguence fragments that overlap slightly in their starting and ending positions e g when using two slightly different primer pairs to extract related seguence 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 Alignment x Select alignment builder Geneious Clustalw A Profile Alianment Cost Matrix 65 similarity 5 0 4 0 y Gap open penalty E Gap extension penalty 3 Alignment type Global alignment Needleman Wunsch y Automatically determine sequences direction gt More Options Cancel Restore defaults Figure 3 9 Options for protein pairwise alignment If you are aligning nucleotide seguences you will also have the option of doing your alignment by translation and back To view the options for translation alignment click the Mor
72. e Options button that the bottom of the alignment dialog The translation alignment options will appear Here you can set the genetic code and translation frame for the translation as well as the cost matrix gap open penalty and gap extension penalty for the alignment If you want to set the alignment type global or local or choose to automatically determine the sequences direction do it in the main section of the dialog 3 5 SEOUENCE ALIGNMENTS 75 Alignment xl Profile Alignment Cost Matrix E F n Gap open penal 128 Gap extension penalty Global alignment Needleman Wunsch v L Figure 3 10 Options for protein pairwise alignment 76 CHAPTER 3 ANALYSING DATA 3 5 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 3 Multiple se
73. e easiest way to trim seguences is at the assembly step Select the trim options you wish to use in the Assembly options and click OK The seguences will be trimmed and assembled in one operation This means you cannot view the trimming that Geneious uses before assembly is performed but the trimmed regions will still be available and adjustable after assembly is complete Trimmed annotations are ignored when calculating the consensus seguence for a contig So although the trimmed regions are visible they do not affect the results of assembly at all Seguence trimming can be performed before assembly by selecting the seguences you wish to trim and selecting Tools TrimEnds This will add Irimmed annotations to the se quences which are ignored in the construction of a contig When performing Assembly from seguences which have been annotated in this way select Use Existing Trim Regions Trimmed annotations can also be created manually using the annotation editing in the seguence 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 Methods used for automatic trimming There are three types of automatic trimming available in Geneious e Trim by error probability is available for chromatogram documents which have quality val
74. e 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 annotations The fragment resulting from digesting 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 Insert into Vector 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 126 CHAPTER 10 CLONING PRO ONLY Insert into Vector Insert Insert forward 0 Insert reverse 689 693 1793 1797 forward strand AATTC reverse strand G Vector Polylinker region to cut within Annotation multiple cloning site Bases to inclusive Entire sequence Candidate Enzymes Enzymes annotated on insert NotI EcoRI Enzyme set Cut vector with EcoRI and NotI w Product insert index 689 693
75. ear as columns in the Document Table These new columns can be used to order the table Take the example of protein size A click on the column heading will order the documents in increasing or decreasing order according to their protein size Clicking the column heading again arranges the documents in the opposite order An arrow next to the heading indicates if it is in increasing A or decreasing V order 2 9 PREFERENCES 49 2 9 Preferences You can access the preferences screen in two ways 1 Shortcut keys Ctrl Shift P Windows Linux Command Shift P Mac OS X 2 Select the Tools Menu and click Preferences There are several sections in the preferences window which are presented as tabs The most important of these are described below 2 9 1 General This contains connection settings data storage details for your local documents automatic new version checking and a Search History Check for new version of Geneious Enable this to have Geneious check for the release of new versions everytime it is started If a new version has been released Geneious will tell you and give you a link to download it Also check for beta version of Geneious Enable this to also have Geneious alert you when new beta versions are released 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 Max memory available to Geneious
76. eious for publication we request that you cite primers 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 at http fokker wi mit edu primer3 Further information on the functionality of the primer design feature can be found in the primer3 documentation available here http frodo wi mit edu cgi bin primer3 primer3_www_help cgi Please note that some controls have been changed renamed or removed from Geneious but most of the primer3 functionality is available 3 8 Contig Assembly Pro only Contig assembly or sequence 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 multiple 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
77. enBank EMBL DDBJ PDB seguences no EST STS GSS or HTGS sequences genome Genomic entries from NCBI s Reference 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 wgs A database for whole genome shotgun sequence entries env nt This contains DNA seguences from the environment i e all organisms put together Table 2 3 Protein seguence searches in the BLAST databases Database Protein searches env nr Translations of seguences 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 Pat
78. ent division of GenBank PDB Sequences derived from 3D structure Brookhaven PDB RefSeq RefSeq protein sequences from NCBI s Reference Sequence Project SwissProt Curated protein sequences information from EMBL protein sequence database You could use this option to find potential translation prod ucts of an unknown nucleotide sequence e tblastn Compares a protein query sequence against a nucleotide sequence database dy namically translated in all reading frames e tblastx Compares the six frame translations of a nucleotide query sequence against the six frame translations of a nucleotide sequence database Please note that the tblastx program cannot be used with the nr database on the BLAST Web page because it is too computationally intensive You can quickly and easily BLAST a sequence document against any of the available BLAST programs via the NCBI Blast menu This can be accessed by selecting a sequence document and going to the Tools menu or by right clicking Ctrl Click on Mac OS on a sequence document Geneious also allows you to specify most of the advanced options that are available in BLAST 2 5 STORING DATA YOUR LOCAL DOCUMENTS 37 To access the advanced options click the More Options button which is by the Search but ton in all NCBI BLAST services Geneious will now display a large text box labelled Search For in which you can enter your guery seguence this will be automatically filled in if you en t
79. ents for seguences in UniProt which match the domain architecture of your Pfam seguence document ie they have the same domains in the same places This operation can take a long time e Get Domains in Seguence creates a domain document for every domain in a Pfam se guence document e If your domain document is a member of a Pfam clan you can use Get Clan to get a document representing that clan e Get Domains in Clan will do the opposite ie get documents representing each domain in a clan e If your domain document contains the seed alignment for the domain you can use Get Full Alignment to get a domain document with the full alignment e Conversely you can use Get seed alignment to get a domain document with the seed alignment only from a domain document with the full alignment e Get Full Seguences will return the full UniProt seguence documents from which the seguences in the alignment in a domain were extracted e Get Full Seguence will return the full UniProt seguence document from which a se guence taken from an alignment in a domain was extracted 108 CHAPTER 6 PFAM PRO ONLY Chapter 7 Smart Folders Pro only Smart folders are a new feature of Geneious that allow you to separate relevant data from extraneous search results retrieved by an agent Smart Folders are created from within the Create Agent dialog To open the Create Agent dialog choose the Agents button from the toolbar and then select
80. er 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 In the future Geneious may support per account options for sharing your documents or even organize contacts into groups so that you can share your documents with specific groups only 9 4 Browsing Searching and Viewing Shared Documents Folders that your contacts have shared will appear beneath that contact just as they do in your contact s own Services panel You can browse these folders as you do your local folders You 9 5 CHAT 119 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 There are also Download item
81. eral method applicable to the search for similarities in the amino acid sequence of two proteins J Mol Biol 48 1970 no 3 443 53 72 73 79 94 17 C Notredame DG Higgins and J Heringa T coffee A novel method for fast and accurate multiple sequence alignment J Mol Biol 302 2000 no 1 205 217 25 18 RJ Roberts T Vincze J Posfai and D Macelis Rebase enzymes and genes for dna restriction and modification Nucl Acids Res 35 2007 D269 D270 121 19 F Ronquist and JP Huelsenbeck Mrbayes 3 Bayesian phylogenetic inference under mixed models Bioinformatics 19 2003 no 12 15724 80 20 N Saitou and M Nei The neighbor joining method a new method for reconstructing phyloge netic trees Mol Biol Evol 4 1987 no 4 406 25 80 83 21 TF Smith and MS Waterman Identification of common molecular subsequences Journal of Molecular Biology 147 1981 195 197 72 73 22 K Tamura and M Nei Estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees Mol Biol Evol 10 1993 no 3 512 526 82 23 JD Thompson TJ Gibson F Plewniak F Jeanmougin and DG Higgins The clustal x windows interface flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 25 1997 no 24 4876 4882 23 25 27 76 78 24 JD Thompson DG Higgins and TJ Gibson Clustal w improving the sensitivity of
82. ered a seguence in the basic search then clicked More Options Below the search box are all of the advanced 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 NCBI BLAST documentation at http www ncbi nlm nih gov blast blastcgihelp shtml If you have a mirror of the NCBI BLAST databases you can set Geneious to use this by select ing the NCBI BLAST service and then clicking the Change database location button and entering the url for the mirror 2 5 Storing data Your Local Documents Geneious can be used to store your documents locally Under the Local folder in the Services Panel you are able to create sub folders to organize and store a variety of document types 2 4 Table 2 4 Geneious document types Document type Geneious Icon Nucleotide seguence Protein seguence Phylogenetic tree 3D structure ALARA Sequence alignment w S A gt gt f Chromatogram Journal articles PDF PEES Other documents 38 CHAPTER 2 RETRIEVING AND STORING DATA This is also where you can set up special folders to receive documents that are downloaded by a Geneious agent To create a new folder in Geneious select the Local folder or a sub folder icon in the services panel and right click Ctrl Click on MacOS This will pop up a menu Clicki
83. erlinks between the pages and external hyperlinks beginning with http will be opened in the user s browser If you want to include figures and diagramsin the pages just put the image files in the 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 folder as above 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 seguence fasta gt click here lt a gt To open more than one document from a link separate the filenames with the pipe character for ex ample lt a href seguence fastalseguence2 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 Once you have all your files together put the contents of the folder in a zip file with the exten 111 112 CHAPTER 8 GENEIOUS EDUCATION PRO ONLY sion t
84. es Phrap Consed PileUp msf Alignments pileup gcg PIR NBRF pir Sequences alignments NBRF PIR Raw sequence text seq Sequences Any file that contains only a sequence Rich Sequence Format rsf Sequences alignments GCGs NetFetch Sequence Chromatograms ab1 scf Raw sequencing trace amp sequence Sequencing machines Vector NTI sequence gb gp Nucleotide amp protein sequences Vector NTI Vector NTI AlignX alignment apr Alignments Vector NTI AlignX Vector NTI Archive mad pa4 0a4 ea4 ca6 Nucleotide amp protein sequences enzyme sets and publications Vector NTI seg2 P GDASYFHATCDSGDGRGGAO0APHKCRCDG ANVVPTYFDYVPOFLRWPEE seg3 KLSNASYFRATC SDGOSGAO0ANNYCRCNGDKPDDDKP NTDPPTYFDYVPOYLRWSEE seq4 DKGNA YFRRTCNSADGKSOSOARNOCRC KDENGKN ADOVPTYFDYVPOYLRWSEE seq5 DKGNA YFRATCNSADGKSOSOARNOCRC KDENGXN ADOVPTYFDYVPOYLRWSEE seg6 P GNAO0YFRNACS EGKTATKGKCRCISGDP PTYFDYVPOYLRWSEE seq7 DNA Star files P KGANYFVYKLD DNAStar seg and pro files are used in Lasergene a seguence analysis tool produced by DNAStar DNA Strider Sequence files generated by the Mac program DNA Strider containing one Nucleotide or Pro tein sequence 2 2 IMPORTING AND EXPORTING DATA 25 EMBL UniProt Nucleotide seguences from the EMBL Nucleotide Seguence Database and protein seguences from UniProt the Universal Protein Resource EndNote 8 0 XML format EndNote i
85. ete 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 ney lt a b C extract F Reverse Complement 3 Translate ED Annotation f Tools Save Consensus CTGG its ad i A 400 410 modified 1 4 T AA CA A Moda amem m c 17 A E A A A A A A A a Figure 3 19 Highlight disagreements and edit to resolve them 3 8 6 Saving 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 3 9 Results of analysis All analysis results are deposited in the currently selected folder If no local folder is selected then you will be prompted for a local folder This applies to sequence alignments phylo genetic trees sequence translations reverse complements and extraction of sequences Once generated analysis results can be dragged to another location if desired 98 CHAPTER3 ANALYSING D
86. eted area By holding down the alt key while deleting residues on the left will be moved into the deleted area instead After editing is complete click Save to permanently save the new contents 62 CHAPTER3 ANALYSING DATA The Pop up menu in the seguence viewer The toolbar actions are available via a pop up menu as well Right click Ctrl click on Ma cOS on any seguence partly highlighted seguence or annotation to show the various options The pop up menu contains the Copy residues action keyboard Ctrl C to copy the selected residues to the system clipboard Printing a seguence view To print a sequence view go to File Print and click OK The view is printed without the options panel It is recommended 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 3 1 2 Dotplot viewer This is a special viewer that appears when two sequences are chosen A dotplot compares two sequences to find regions of similarity Each axis X and Y on the plot represents one of the sequences being compared Figure 3 4 For more information on dotplots see section 3 4 Chicken 1737 nt W Zoom Level W 1000 28 T Ss AR KA y Preferences a Stringency 18 a gt 4 x Window size 21 1000 Australobius 1815 nt yi j
87. f the residue value at that position and the values on either side Chromatogram This is available with chromatogram traces It displays the four traces above the sequence where the peak as detected by the base calling program is at the middle of the base letter When viewing more than one chromatogram or an alignment made from chro matograms each chromatogram can be turned on or off individually using the checkbox s below 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 Quality This is available with enabled chromatogram traces It 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 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 10 probability of calling error while the middle represents a probability of only a one in a thousand 1073 Coverage This is available on sequence alignments and contigs The height of the graph at each position represents the number of sequence which have a non gap character at that position If the selected contig was created using Geneious and it contains sequences in both directions then color coding is used to indicate whether each position is covered by reads in both
88. factors stringency and window size The strin gency is the number of matches reguired in the given window size for a dot to be plotted and it acts as a threshold that determines the sensitivity of the dot plot Reducing the reguired num ber of matches for a given window size will increase the sensitivity of the dotplot but will also lead to more false positives regions that match due to chance alone If the stringency is set to 3 and the window size to 5 then a pair of 5 base windows that contain 3 or more matching nucleotides will be classified as a match A dot corresponding to the center of the two windows will then be displayed in the dotplot Anything less will be classified as a mismatch and no dot will be drawn 3 4 1 Viewing Dotplots To view a dotplot in Geneious select two nucleotide or protein sequences in the Document Table and select Dotplot Viewer in the Document Viewer Panel Figure 3 4 The Dotplot Viewer allows you to zoom in and out and to customize the stringency and window size setting If a single nucleotide or protein sequence is selected then the dotplot is also available In this case it shows a comparison of the sequence to itself The dotplot comparison of two sequences is drawn from top left to bottom right in colors rang ing from blue slight similarity to red perfect similarity The dotplot in Geneious also simul taneously draws the comparison of one sequence to the reverse complement of the other This
89. g3 KRIYKKIFKEIHSGLSTKNGVKDRYON DGDNYFOLREDWWTANRSTVWKALTCSD seq4 SORHYKD DGGNYFOLREDWWTANRHTVWEAITCSA seas NVAALKTRYEK DGONF YOLREDWWTANRATIWEAITCSA seg6 FSKNIX O0IEELODEWLLEARYKD TDNYYELREHWWTENRHTVWEALTCEA seq7 KELWEALTCSR seql GGGKYFRNTCDG GONPTETONNCRCIG ATVPTYFDYVPOYLRWSDE 24 CHAPTER 2 RETRIEVING AND STORING DATA Format Extensions Data types Common sources Clustal aln Alignments ClustalX DNAStar seg pro Nucleotide amp protein seguences DNAStar DNA Strider str Seguences DNA Strider Mac program ApE Embl UniProt emb swp Seguences Embl UniProt Endnote 8 0 XML xml Journal article references Endnote Journal article websites Fasta fasta fas etc Sequences alignments PAUPX ClustalX BLAST FASTA 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 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 assembli
90. gging 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 To quickly select a single residue double click on it Triple clicking will select a block of residues within a single sequence Quadruple clicking selects a block of residues in multiple sequences The shift and control alt on a Mac keys can be combined with the keyboard arrow keys to select sequence and alignment regions The shift key extends the current selection and holding down the control alt on a Mac key while pressing the keyboard arrow is equivalent to pressing it 10 times These can be used together For example in an alignment if you have a region of one sequence selected and would like to select the same region in all sequences then you could press control up until you reach the first sequence and then press control shift down and few times until all sequences are selected 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 MacOS on the sequence name and choosing the remove sequence option Alternatively select the entire sequence by clicking on the sequence name and press the delete key To delete a region of an alignment select the region and press the delete or backspace key Normally this will move residues on the right into the del
91. guence of fixed length because YS matches CC CG TC and TG On a random seguence with uniform nucleotide distribution it would match approximately once every nucleotide as would a recognition seguence of length 1 hence the effective length of YS is 1 e Only include enzymes that match X to Y times lets you filter the results once the restriction sites have been identified If checked this option will discard all restriction sites for en zymes whose recognition seguence matches less than X or more than Y times If you set X to be 0 when this operation is complete it will report which candidate enzymes matched 0 times 10 2 DIGEST INTO FRAGMENTS 123 e Exclude enzymes cutting between residues lets you annotate only enzymes which do not cut within a certain range e If you select to show More Options a table of all enzymes in your candidate set filtered by the effective recognition seguence length constrained when active will be displayed 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 If not all candidate enzymes are currently selected because of a recognition seguence length constraint or because you have selected a subset of the table rows yourself you can sa
92. h enzymes to look for on the vector sequence 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 128 CHAPTER 10 CLONING PRO ONLY 10 3 3 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 1f th
93. h graph controls the height of that graph A number of graphs are available Similarity This is available for sequence alignments It displays the similarity across all se quences for every position Green means that the residue at the position is the same across all sequences Yellow is for less than complete similarity and red refers to very low similarity for the given position Figure 3 2 OI gt Extract 9 Reverse Complement amp Translate P Edit MP Annotation Save O A1 KE 00 K GTAGGATGGC CTGAGGTTAG GGAAAGAATG ii it A1 TZ 97 GTGGGATGGC CTGAGATTAG GGAAAGAATG v M Graphs A SN 96 D GCGGGATGGC CTAAGGTTAG GGATAGGATG M similarity A1 UG 99 GTGGGATGGC CTAGGGTTAG GGAAAGAATA Siting indio 18 A1 KE 00 K AAGGGATGGC CTGAAGTTAG GGAGAGAATG Similarity N Ee a gt Consensus A1 KE 00 K AGACGTGCTC GTGCTCCTGC AGCAGCACCT Annotations A1 TZ 97 AGACGAACTC nono gt Layout A SN 96 D AGACAAACTC CTCCA gt M Colors A1 UG 99 AGACAAACTC CT gt W Zoom Options A1 KE 00 K AGRAATACCE asus SN Oo s8 p Statistics 636 85 Similarity nii mun ma 100 110 120 A1 KE 00 K ACTGCCGCAG CACCTGGAGT AGGAGCAGTT A1 TZ 97 CTACAGCAG CAACAGGAAC AGGAGCAGTA A SN 96 D ACACCAGCAG CAAAAGGAGT AGGAGCAGTA A1 UC 99 CCAGCAG CAGAAGGAGT AGGAGCAGCA A1 KE 00 K CTGCACCAG CACCAGGAGT AGGAGCAGTA Similarity mE ZL iin Nl n Alt left click
94. he Document Table has some useful 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 Column 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 MacOS 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 MacOS 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 remain hidden in all areas of t
95. he Menu bar File Menu This contains some standard File menu items including printing and Exit on Windows It also contains options to create rename delete share and move folders and Import Export options Edit Menu Here you will find common editing functions including Cut Copy Paste Delete and Select All These are useful when transferring information from within documents to other locations or exporting them This menu also contains Find in Document Find Next and Find Previous options Find can be used to find text or numbers in a selected document This is useful when looking for annotated regions or a stretch of bases in a sequence This opens a Find Dialog The shortcut to this is Ctrl F Next finds the next match for the text specified in the Find dialog The shortcut keys are F3 or Ctrl G Geneious then allows you to choose another document and continue searching for the same search word Prev finds the previous match The shortcut keys for this are Ctrl Shift G or Shift F3 View Menu This contains several options and commands for changing the way you view data in Geneious e Back Forwards and History allow you to return to documents you had selected previously e Search is discussed in section 2 3 e Agents are discussed in section 2 6 e Next unread document selects the next document in the current folder which is unre
96. he program until you show it again 18 CHAPTER 2 RETRIEVING AND STORING DATA 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 e 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 your self however e Save Column 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 If a Note is added to a document refer to the section on Adding Notes for more in formation a Note column is added to the end of the existing Document table Also when accessing BLAST 1 in Geneious the Document Table has additional columns related to the BLAST search 2 1 3 The Document Viewer Panel The Document Viewer Panel shows the contents of any document clicked on in the Document Table To view large documents it is sometimes better to double click on them This opens a view in a new window In the document viewer panel there are
97. ignments for annotations which match certain criteria then extract all of the matching annotations to separate sequence documents Includes the option to concatenate all matches in each sequence into one sequence document Useful for extracting a certain gene from a group of genomes e Strip Alignment Columns creates a new alignment document with some columns for example all identical columns or all columns containing only gaps stripped e Concatenate Sequences or Alignments Joins the selected sequences or alignments end on end creating a single sequence or alignment document from several After selecting this operation you are given the option to choose the order in which the sequences or alignments are joined You can also choose whether the resulting document is linear or circular and 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 e Generate Consensus Sequence Generates a consensus sequence for the selected se quence alignment and saves it to a separate sequence document After selecting this operation you are given options for choosing what type of consensus sequence you wish to generate see section 3 1 1 for more details on the options 22 CHAP
98. in the NCBI genetic database Each organism must be represented by at least one nucleotide or protein seguence 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 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 scope and depth of these databases make them critical information sources for molecu lar biologists and bioinformaticians alike However a library is only as good as its librarian Geneious is your librarian allowing you to search for filter and store only the data that you care about 2 4 4 Accessing NCBI BLAST through Geneious BLAST 1 stands for Basic Local Alignment Search Tool It allows you to query the NCBI sequence databases with a sequence in order to find entries in the public 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 The result of a BLAST query is a tab
99. is box is checked a document will be created representing the fragment removed from the vector if any If the insert fragment was produced from a seguence with two restriction site annotations the fragments on either side of the restriction site annotations will also be kept Chapter 11 Server Databases Pro only By using server databases Geneious can store your documents in your favorite relational SOL database rather than on the file system This means that multiple users can concurrently use the same synchronized storage location without any problems A server databases can be used for everything a local database is used for This includes col laboration and smart agents 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 11 1 Supported Database Systems To use a database as a server database Geneious reguires that it support transactions with an isloation level set to SERIALIZABLE Supported databases systems include Microsoft SOL Server PostgreSOL Oracle and MySOL It is possible to use other database systems if you provide the database driver see section 11 2 1 Server Databases have been tested using Microsoft SOL Server 2005 Express PostgreSOL 7 4 Oracle 10g Express Edition MySOL 5 129 130 CHAPTER 11 SERVER DATABASES PRO ONLY 11 2 Setting u
100. is drawn from bottom left to top right in colors ranging from green slight similarity to yellow perfect similarity 3 4 2 Interpreting a Dotplot e Each axis of the plot represents a sequence e A long 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 e 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 amp Lenk 14 72 CHAPTER 3 ANALYSING DATA 3 5 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 i e 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 evoluationary path to one descendant sequence this sequence will show a special gap character
101. ith 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 The sequence logo graph has an option to Weight by quality This is very useful for identifying low quality regions and resolving conflicts lt Extract G Reverse Complement Translate P Edit MP Annotation 4 Tools El Save Consensus ae W 1 N 62 19 3 2 4 Ka i i Figure 3 18 The overview of a contig Finding disagreements or SNPs To easily identify bases which do not match the consensus turn on Highlight Disagreements in the consensus section of the seguence viewer options When this is on any base in the seguences which matches the consensus at that position is grayed out and bases not matching are left colored With this on you can guickly jump to each disagreement by pressng Ctrl D Cmd D on Mac OS or by clicking the Next Disagreement button in the seguence 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 reference seguence and you are interested in finding differences between each sequence and the reference or SNPs 3 9 RESULTS OF ANALYSIS 97 3 8 5 Editing Contigs Editing a contig is exactly the same as editing an alignment in Geneious After selecting the contig click the Edit button in the sequence viewer and you can modify insert and del
102. ity sorting The Filter allows you to instantly 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 The Sort button in the toolbar provides two actions in a popup menu Sort by similarity is available when a single seguence document is selected in the Document Table It will rank all other seguences by their similarity to the selected seguence The most similar seguence is placed at the top and the least similar seguence at the bottom This also produces an E value column describing how similar the seguences are to the selected one The Remove Sort by Similarity action will remove the E value column and return the table to its previous sorting 2 71 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 2 8 NOTES 45 2 8 Notes Notes allow you to add arbitrary information to
103. le of hits Each hit refers to a GenBank accession number and the gene or protein name of the sequence Each hit also has a Bit score which provides information about how similar the hit is to the query sequence The bigger the bit score the better the match Finally there is also an E value or Expect value which 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 Geneious is able to run NCBI BLAST on many different databases Some of these databases are non redundant in order to reduce duplicate hits You can submit either a raw sequence or Genbank accession number into NCBI BLAST and receive a summary of results for each hit This summary contains the bit score e value identity and the stretch of the query sequence and hit sequence that match The databases that can be searched are Geneious can perform five different kinds of BLAST search e blastp Compares an amino acid query sequence against a protein sequence database e blastn Compares a nucleotide query sequence against a nucleotide sequence database e blastx Compares a nucleotide query sequence translated in all reading frames against a 36 CHAPTER 2 RETRIEVING AND STORING DATA Table 2 2 Nucleotide seguence searches in the BLAST databases Database Nucleotide searches nr All non redundant G
104. ll pairs of bases at the same column and scoring a hit one when they are identical divided by the total number of pairs Identical sites The number of sites that are identical across all seguences Annotations Some protein and nucleotide seguences come with annotations and these can be viewed within Geneious sequence viewer In the presence of annotations the options panel includes an An notations check box Figure 3 3 Uncheck the check box to turn off all annotations Individual annotation types can be turned on or off by using the check boxes next to them Compress annotations This option reduces the vertical height of the annotations on display This reduces the space occupied by annotations by allowing them to overlap and increases the amount of the seguence displayed on the screen Hide all Show all These buttons can be used to turn off and on all annotations on the seguence The seguence viewer toolbar The top of the seguence viewer panel shows a toolbar containing several actions Some of them operate on a part of a seguence or alignment There are several ways to make such a selection 60 CHAPTER3 ANALYSING DATA Sequence View Text View Notes Extract Sf Reverse Complement ES Translate X Edit MP Annotation M Save 549172 MRMESRARSA CTWISCTSES PCSTSCPPSP AMPTEERRRS HPOORRRPSS SPNRRURGWT TSPCPTRSEM KILOT KEN Transmembrane protein HWLF3 Z SS gt Graphs US20 gene v M Annota
105. lude 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 pro grams such as PHYLIP PAUP ClustalW 24 ClustalX 23 Tree Puzzle 8 and PROTML Geneious is also able to read trees in Newick format and display them in the visualization win dow It also gives you a number of display options including tree types branch lengths and labels Nexus format The Nexus format 13 was designed to standardize the exchange of phylogenetic data in cluding seguences 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 PDB format 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 an interactive 3D view of the protein structure including support for displaying the protein s secondary structure when the appropriate information is available PDF format PDF stands for Portable Document Format and is developed and distributed by Adobe Systems http www adobe com It
106. m 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 freguent topology will be added and so on until the total freguency of the topologies reaches the threshold value 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 3 6 8 Tree building in Geneious Geneious can build a phylogenetic tree for a set of sequences using pairwise genetic distances 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 Tree building from an alignment If you are building a tree from an alignment the following options are seen in the tree window If you select a tree document which contains an alignment then the alignment will simply be extracted from the tree and used in the tree building process 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 seque
107. nces button allows you view the selected nodes in the sequence viewer The Root button allows you to re root the tree on the selected node The Swap Siblings button allows you to swap the position of the sibling clades of the selected node 3 1 5 The Chromatogram viewer This viewer is hidden by default in Geneious To turn it on select Tools Preferences then enable it on the Plugins tab The Chromatogram viewer provides a graphical view of a the output of a DNA sequencing machine such as 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 processed 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 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 3 2 LITERATURE 69 largest base represents a one in a million 10 or smaller probability of calling error while half of that represents a probability of only a one in a thousand 10 Mark calls Draw a vertical line showing the exact location of the call m
108. nces you only have the option of Jukes Cantor distance correction e Tree building method There are two methods under this option Neighbor joining 20 and UPGMA 15 e Create consensus via resampling Check this box to build a consensus tree using resampling of sequence alignment data e Resample tree Check this to perform resampling 84 CHAPTER3 ANALYSING DATA e O e Tree Select tree builder Geneious PE Consensus f PHYML Genetic Distance Model Tamura Nei Tree build Method Neighbor Joining Outgroup No Outgroup Bl Consensus Tree Options Resample tree Resampling Method Jackknife gt Number of samples 1 000 8 Create Consensus Tree Sort Topologies Support Threshold 50 8 o E OJO Topology Threshold C Save raw trees a OD A Figure 3 12 Tree building options in Geneious 3 7 PCR PRIMERS PRO ONLY 85 e Resampling method Either bootstrapping or jackknifing can be performed when resam pling columns of the seguence 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 samples e Sort Topologies Produce trees which summarise the topologies resulting from resampling See above for more details Support threshold This is used to decide which monophyletic clades to include in the con sensus tree af
109. ng 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 messages 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 9 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 in case it is not sufficient for you that the communication with our 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 Wildfire from Ig nite Realtime http www igniterealtime org projects wildfire index jsp which is available for free under the GNU General Public License
110. ng on New folder opens a dialog that will prompt you to name the folder The named folder is then created as a sub folder of the folder that you originally right clicked on Important Search results will be lost when you exit Geneious unless the downloaded docu ments have been copied or moved to one of your local folders In Geneious you can create new folders rename existing folders delete and export folders All these choices are available by either right clicking on the folder clicking on the action menu Mac OS X or by holding down the control button and clicking Mac OS X Also 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 2 5 1 Transferring data It is guick and easy to transfer data to your local folders from either a Geneious database search or from your computer s hard drive Please check you have already set up your destination folders before continuing Moving documents from Geneious searches to your Local folders There are a number of ways to do this Drag and drop This is guickest 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 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 N
111. ng stored on your hard drive open the Tools menu in the Menu Bar Click Tools Preferences General Your documents are stored at the location specified by the Data Storage Location field see Figure 2 8 You can change this location by clicking the Browse button and selecting a new location Geneious will remember this new location when you exit en Preferences o L General Plugins o General settings Data Storage Location Users alexei Geneious Browse Figure 2 8 Setting the location of your local documents 2 6 AGENTS 41 2 6 Agents Geneious offers a simple way for you to continuously receive the latest information on genomes seguences and protein structures This feature is called an agent Each agent is a user defined automated search You can instruct an agent to search any Geneious accessible database at reg ular intervals eg weekly including your contacts on Collaboration This simple but powerful feature ensures that you never miss that critical article or DNA seguence To manage agents click on the agent icon in the toolbar An agent has to be set up before it can be used 2 6 1 Creating agents To set up an Agent click the Agents icon and the create button You now need to specify a set of search criteria in the exact same way as you do for search the database to search search freguency and the folder you wish the agent to deliver its results to The sea
112. o selection If this option is turned on when you select a range of sequence residues the seguence viewer automatically zooms in or out so that the selected piece fills the entire viewing area A shortcut is to select the zoom options checkbox without expanding it 3 1 DOCUMENT VIEWERS IN GENEIOUS 59 Default zoom level This options allows the user to specify the initial level of zoom when viewing a seguence Please note Geneious automatically restores the previous zoom level and over rides the de fault settings when you return to a seguence that you were previously viewing Statistics This displays some statistics about the seguence being viewed They correspond to the se guence alignment being viewed or the highlighted part of the seguence alignment The length of the seguence or part of the seguence is displayed next to the Statistics option Residue freguencies This section lists the residues for both DNA and amino acid seguences and also for alignments It gives the freguency of each nucleotide or amino acid over the entire length of the seguence including gaps If there are gaps then a second percentage freguency is calculated ignoring gap characters The G C content for nucleotide seguences is shown as well for easy reference The following statistics are available when viewing alignments or multiple seguences Pairwise Similarity The average percent similarity over the alignment This is computed by looking at a
113. o shift hold ctrl right mouse button and drag Figure 3 5 A view of a 3D protein structure in Geneious You can customize your view of the 3D structure in several ways Position e Spin makes the molecule spin around the y axis e Center on click will centre the molecule on any atom you left click on e Reset position resets the position of the molecule to where it started Show Atoms Check this box to show the atoms in the molecule The following settings apply 64 CHAPTER3 ANALYSING DATA e Atom size is expressed as a percentage of the Van Der Waals radius e Show hydrogens toggles the visibility of hydrogen atoms e Show atom symbols toggles the visibility of atomic symbols for each atom Bonds e Show bonds toggles the visibility of bonds between atoms e Show H bonds toggles the visibility of hydrogen bonds between atoms The size of the bonds is expressed as their radius in ngstr m Show backbone Check this box to highlight the molecule s backbone a part of its secondary structure e Backbone type changes the way the backbone is rendered e Backbone size changes the rendering size of the backbone in A Measure distances When this box is checked you can measure the distance between two atoms by left clicking on both of them Measurement units changes the units of the measurements Colors e Atoms Lets you choose a color scheme for atoms and bonds e Backbone Lets you choose a color scheme
114. o 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 with any existing Jabber service such as Google Talk but we recom mend using the Geneious default t alk geneious conm 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 9 5 3 This chapter shows you how to e Create a new collaboration account e Search for and add contacts to your account e Share local folders with your contacts Search your contacts as you would an online database Set up and run your own Jabber server 9 1 Managing Your Accounts When you start Geneious you will see the empty Collaboration service in the Services Panel and the Collaboration menu at the top You can open the Add New Account dialog by either 113 114 CHAPTER 9 COLLABORATION PRO ONLY right clicking Ctrl click on MacOS on Collaboration in the Services Panel and clicking Add New Account in the popup menu or by selecting the same option from menu at the top 9 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
115. ore all your local documents note types and program preferences A file in Geneious format will usually have a geneious extension ora xml extension This format is useful for sharing documents with other Geneious users and backing up your Geneious data Geneious Education format 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 8 for information on how to create such files MEGA format 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 e 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 cml format files in Chemical Markup Language e gpr format ghemical files hin format files produced by HyperChem e nwo format files produced by NWChem Newick format 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 2 2 IMPORTING AND EXPORTING DATA 27 taxa separated by a comma Some trees inc
116. ournal articles sequence alignments and trees By clicking on the search icon you can search data for text or by sequence similarity BLAST You can enter a search string into the Filter box located at the right side of the toolbar this will hide all documents that do not contain the search string For more information see section 2 1 2 1 2 3 The Document Viewer Panel The Document Viewer Panel is where sequences alignments trees 3d structures journal ar ticle abstracts and other types of documents can be shown graphically or as plain text Many document viewers allow you to customize settings such as zoom level color schemes layout and annotations nucleotide and amino acid sequences three different layouts branch and leaf labeling tree documents and many more When viewing journal articles this panel in cludes direct link to Google Scholar All these options are displayed on the right hand side of the panel Figure 1 2 For more information see section 2 1 3 O exec G reverse complement G Transie 4 eat Y arnoraon Ms AOSAMCAGCA emCAMCECEA ECATCERATC CACTAGCTAG CTAGCTAGCT i imating mutation parameters population history and genealogy simultaneously from temporally spaced sequence Alexi J Drummond Geoff K Nicholls Allen G Rodrigo amp Wiremu Solomon MCECATGACT GRTGACTAEC TACGATCGAT CGATCGACTG ACTERCTORC TGACTAGCTC AGATCGATCG AICGATCGAT CGATCGACTG ACTGACTGAC TGACTEACTE ACTCAGATCG ATC
117. ous provides some basic phylogenetic tree reconstruction algorithms for a preliminary in vestigation of relationships between newly acguired seguences For more sophisticated meth ods of phylogenetic reconstruction such as Maximum Likelihood and Bayesian MCMC we recommend specialist software such as MrBayes 19 and PhyML 7 which are available as a plugins to Geneious These can be downloaded from the plugins page on our website Geneious implements the Neighbor joining 20 and UPGMA 15 methods of tree reconstruc tion 3 6 1 Phylogenetic tree representation A phylogenetic tree describes the evolutionary relationships amongst a set of seguences They have a few commonly associated terms that are depicted in Figure 3 7 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 seguence alignment Nodes or internal nodes of a tree represent the inferred common ancestors of the seguences that are grouped under them Tips or leaves of a tree represent the seguences 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
118. ows in the rare cases when it is needed e Otherwise if you select Digest using Enzyme Set the digestion operation includes finding the restriction sites first but without generating the annotations Therefore the options are the same as for Find Restriction Sites which is discussed in section 10 1 124 CHAPTER 10 CLONING PRO ONLY Find Restriction Sites Candidate Enzymes Commercially Available Enzymes 632 W Minimum effective recognition sequence lenath nucleotides a v _ Only include enzymes that match to times Em Exclude enzymes cutting between residues 234 E 629 enzymes selected Recognition TTA TAA CACCTGC 4 8 GACNNNN NN AGG CCT GACGT C CC TCGAGG TGC GCA ACCTGC 4 8 G GTACC G GYRCC CCANNNN NTGG CCGCTC 3 3 GT MKAC CG CG T CCGGA CCGC 3 1 AA CGTT GGATC 4 5 Y GGCCR R AATTY CTGAAG 16 14 CAC GTG GR CGYC CACNNN GTG GT AC Effective Len Overhang blunt 5 4 nucleotides 3 2 nucleotides blunt 3 4 nucleotides S 4 nucleotides blunt 5 4 nucleotides 5 4 nucleotides 5 4 nucleotides 3 3 nucleotides blunt 5 2 nucleotides blunt 5 4 nucleotides S 2 nucleotides 5 2 nucleotides 5 1 nucleotide 5 4 nucleotides 5 4 nucleotides 3 2 nucleotides blunt 5 2 nucleotides 3 3 nucleotides blunt 1 per match 1 per match 1
119. p 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 to 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 Server Database as intended There are two ways you can use your database with multiple users The simple way is just to use the server 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 11 4 1 Your database should now be ready to use with Geneious Now all users can connect to the database by clicking on Server Databases in the service tree and then clicking Connect to a setup database This will bring up a dialog for the u
120. p ww history of its carnivore host Population genetic estimation of the loss of genetic diversity O Population genetic estimation during horizontal transmission of HIV 1 BMC Evol Biol Charles T T 16556318 http ww J Relaxed phylogenetics and da pss mus s AITA NT TKU PUN PLoS Biol Alexei Dru 16683862 http bic JA modified cc cd11 M13F C05 modified cc cd11 M13F C05 022 ab1 Length 597 GCTCACGA JA cc_cd11_M13F_C05_022 ab1 cc cdll M13F C05 022 ab1 Length 597 gctsacgatgc WA 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 p New nucleotide seguence New nucleotied seguence A new nucleotide seguence entered ACGATCAC K 1996YangGeneticsv144p194 1996YangGeneticsv144p1941 pdf 5 tree txt tree txt 4 tips m s e ja JE tree3 txt tree3 txt 1 Trees o Hldentical Journal Title First Author PMID Seguence Resi URLFR 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 actions available for any particular document or group of documents right click Ctrl click on MacOS on a selection of them Ctrl Click on Mac OS X These options vary depending on the type of document T
121. per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match 1 per match Only consider enzymes with recognition sequence Restriction enzyme information was obtained from rebase a free database 4 Save Selected Enzymes Restore Defaults Fewer Options Figure 10 1 Find Restriction Sites options dialog with extended options showing 10 3 INSERT INTO VECTOR 125 Digest into fragments Digest using O Annotated cut positions 2 Enzyme set enzymes from lab fridge 6 Minimum effective recognition sequence length 3 2 nucleotides to times 6 enzymes selected Recognition Segu Effective Length Overhang 8 0 5 2 eotides 1 per match CCTCAG 2 7 0 eo 1 per match GEAG 8 0 4 nucleotides 1 per match 11 13 CAANNNNNG 7 0 5 2 nucleotides 2 per match GGCCGGACC 8 0 5 4 nucleotides 1 per match RTGCAGCAY 7 0 blunt 1 per match A Fewer Options Restore Defaults Figure 10 2 Digest into fragments options dialog with extended options showing 10 3 Insertinto Vector The option Insert into Vector from the Tools Restriction Analysis menu or the context menu allows you to take an insert and insert it into a vector The insert must be one of th
122. progres sive multiple sequence alignment through sequence weighting position specific gap penalties and weight matrix choice Nucleic Acids Res 22 1994 no 22 4673 4680 23 27 78
123. proved your reguest to do so 9 2 1 Add Contact Select your account in the Services Panel and choose Add Contact from the menu at the top or right click Ctrl click on MacOS on your account in the Services Panel and choose the same option You will see a simple dialog with one field Jabber ID A Jabber ID looks like an email address and has a similar function It uniguely 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 Services Panel and it will appear in a tool tip If the server supports it you should also see a Search For Contact link Click this to go to the next dialog 9 2 MANAGING YOUR CONTACTS 117 Add Contact to myaccount Jabber ID e g user name talk geneious com 2 Search For Contact Figure 9 3 Add Contact dialog box Here you will see a box for a search string and 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 Add Contact to myaccount Username Name v Email A Enter Contact ID Add alecta Figure 9 4 Add New Contact dialog box in searching mode Your new contact will appear immediately in your contact list however you will no
124. ptions Figure 3 16 Primer design advanced options Primer Picking Weights At the bottom of both the advanced primer and DNA probe options 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 param eters 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 92 CHAPTER 3 ANALYSING DATA less likely a primer or probe will be chosen if it does not meet the optimal value Some of the weights allow you to specify a Less Than and Greater Than This is for options which allow you 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 3 7 5 More Information The Primer feature in Geneious is based on the program Primer3 http frodo wi mit edu cgi bin primer3 primer3 www cgi 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 Gen
125. quence 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 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 5 Their algorithm used a guide tree to choose which pair of sequences alignments to align at each step Many variations of the progressive pairwise
126. quences and functions from Swiss Prot TrEMBL and PIR It has three main components each optimized for a particular purpose 2 4 3 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 34 CHAPTER 2 RETRIEVING AND STORING DATA NCBI BLAST searches Table 2 1 Table 2 1 NCBI databases accessible via Geneious Database Coverage Genome Whole genome sequences Nucleotide DNA sequences PopSet sets of DNA sequences from population studies Protein Protein sequences Structure 3D structural data PubMed Biomedical literature citations and abstracts Taxonomy Names and taxonomy of organisms SNP Single Nucleotide Polymorphisms Gene Genes The Entrez Genome database This provides views of a variety of genomes complete chromo somes sequence maps with contigs contiguous sequences and integrated genetic and physi cal maps 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
127. rch freguency 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 e g If you have already read all publi cations by a particular author and you want the agent to only get publications released in the future 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 appropri ately You can do that by navigating to the parent folder in the Deliver to box and click New Folder or by creating a new folder beforehand 1 Right click Ctrl click on MacOS 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 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 Otherwi
128. ree can take a long time since it reguires making a pairwise alignment between each pair of seguences 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 typically speeds up the process that may not be the case when the seguences are very distant genetically Alignment MN x Select alignment builder Geneious Clustalw A Profile Slianment Cost Matrix 65 similarity 5 0 4 0 v Gap open penalty 12 Gap extension penalty 3m Alignment type Global alignment v I Automatically determine sequences direction Refinement options Refinement iterations n 7 Build guide tree via alignment faster gt More Options Restore defaults Cancel Figure 3 11 The multiple alignment window You can also do a multiple alignment via translation and back as with pairwise alignment 78 CHAPTER 3 ANALYSING DATA 3 5 3 Sequence alignment using ClustalW Pro only ClustalW is a widely used program for performing sequence alignment 24 23 If you have ClustalW installed your computer Geneious pro allows you to run ClustalW directly from inside the program without having to export or import your sequences If you do not have ClustalW or are unsure if you do you should attempt to perform a ClustalW alignment without
129. ree in the viewer Circular and Unrooted views e Angle Range Compresses the branches into an arc Circular view only Formatting There are a range of formatting options Transform branches allows the branches to be equal like a cladogram or proportional Leaving it unselected leaves the tree in its original form Ordering orders branches in increasing or decreasing order of length but within each clade or cluster 3 1 DOCUMENT VIEWERS IN GENEIOUS 67 Show root branch displays the position of the root of the tree has no effect in the unrooted layout Line weight can be increased or decreased to change the thickness of the lines representing the branches Auto subtree contract automatically contracts subtrees when there is not enough space on screen to display them nicely Show selected subtree only shows only the part of the tree that is selected or the entire tree if there is no selection If you are unfamiliar with tree structures please refer to Figure 3 7 for the following options Root Branch Node Tip Figure 3 7 Phylogenetic tree terms Show tip labels This refers to labels on the tips of the branches of the tree Show node labels This refers to labels on the internal nodes of the tree Show branch labels This refers to the branches of the tree Fach of the three above options has fields that you can set to customise what the labels display e Display allows you to select what information the label
130. rom there into a local folder of your choice Important if you don t drag the documents from a database search into your local folders the results will be lost when Geneious is closed 2 2 4 Data output formats Each data type has several export options Any set of documents may be exported in Geneious native format Data type Export format options DNA sequence FASTA Genbank XML Genbank flat Geneious Amino acid sequence FASTA Genbank XML Genbank flat Geneious Protein 3D structure PDB FASTA Geneious Multiple sequence alignment Phylip phy FASTA NEXUS 13 MEGA3 12 Geneious Phylogenetic tree Phylip phy FASTA NEXUS 13 Newick MEGA3 12 Geneious PDF document PDF Geneious Publication EndNote 8 0 Geneious Additionally documents imported in any chromatogram or molecular structure format can be re exported in that format as long as no changes have been made to the document 30 CHAPTER 2 RETRIEVING AND STORING DATA 2 2 5 Export to comma separated values CSV file The value displayed in the document table can be exported to 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 Please note this format cannot be imported currently 2 3 Searching Searching is designed to be as user friendly as possible and the process is the
131. rs 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 egual to and is less or egual 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 2 13 2 Constraints for constrained string Dy 2 Figure 2 13 The Edit Constraints window 48 CHAPTER 2 RETRIEVING AND STORING DATA Using Note Types The main purpose of Notes is to add user defined information to Geneious documents How ever Notes and Note Types can be searched for and filtered as well Also documents can be sorted according to the values of an added Note Searching Once a Note Type is defined and a Note of that type added it is automatically added to the standard search fields These are listed under the Advanced Search options in the Document Table From then on you can use them to search your Local Documents If you have more than one Field Type for a Note Type they will both appear as searchable fields in the search criteria Filtering Note values can be used to filter the documents being viewed To do so type a value of your Note Type into the Filter Box in the right hand side of the Toolbar Only matching documents will be shown Sorting The fields and values of an added Note Type will app
132. rt 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 Note The above definitions apply to rooted trees The same principles can be applied to un rooted trees by replacing clades 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 3 6 BUILDING PHYLOGENETIC TREES 83 3 6 7 Sorttopologies This will produce one or more trees summarizing the results of resampling The freguency of each topology in the set of original trees is calculated and the topologies are sorted by their freguency 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 fro
133. s Proxy port Config file location GO Cancel Apply Restore defaults Figure 1 6 General Preferences Chapter 2 Retrieving and Storing data Geneious is a one stop shop for handling and managing your bioinformatic data This chapter summarizes the different ways you can use Geneious to acguire update organize and store your data By the end of this chapter you should be able to e Know the purpose of each panel in Geneious Import Export data from various sources Organize your data into easily accessible folders Automatically update your data Know about the advantages of the Note functionality Customize Geneious to meet your needs 2 1 The main window This section provides more information on each of the panels in Geneious Figure 2 1 Figure 2 1 Geneious main window 15 16 CHAPTER 2 RETRIEVING AND STORING DATA 2 1 1 The Services Panel The Services Panel shows a tree that concisely displays sources of data and your stored docu ments 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 Service Panel allows you to access e Your Local Documents e NCBI databases BLAST 1 Genome Nucleotide PopSet Protein Pubmed Structure and Taxonomy e An EMBL database Uniprot e Your contacts Geneious databases You can
134. s a popular reference and bibliography manager EndNote lets you search for journal articles 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 format The FASTA file format is commonly used by many programs and tools including BLAST 1 T Coffee 17 and ClustalX 23 Each seguence 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 seguence data may span multiple lines and these seguence may contain gap characters An empty line may or may not separate consecutive seguences Here is an example of three seguences in FASTA format DNA Protein Aligned DNA gt Orangutan ATGGCTTGTGGTCTGGTCGCCAGCAACCTGAATCTCAAACCTGGAGAGTGCCTTCGAGTG gt gi 532319 pir TVFV2E TVFV2E envelope protein ELRLRYCAPAGFALLKCNDADYDGFKTNCSNVSVVHCTNLMNTTVTTGLLLNGSYSENRT OIWOK gt Chicken CTACCCCCCTAAAACACTTTGAAGCCTGATCCTCACTA CTGT CATCTTAA GenBank files Records retrieved from the NCBI webiste http www ncbi nlm nih gov can be saved in a number of formats Records saved in GenBank or INSDSeg XML formats can be imported into Geneious 26 CHAPTER2 RETRIEVING AND STORING DATA Geneious format The Geneious format can be used to st
135. s display Branch Labels have fixed settings but you can select what the Tip Labels display either Taxon Names Node Heights Seguence Names or a number of other options depending on the tree you are viewing If you are viewing a consensus tree you can also display consensus support as a percentage on node labels e You can use Font to change the size of the labels The tree viewer will shrink the font size of some labels if they cannot all fit in the available space Minimum Size specifies the minimum size that the tree viewer is allowed to shrink the label font to 68 CHAPTER3 ANALYSING DATA e Significant Digits sets how many digits to display if the value the node is displaying is numeric Show scale bar This displays a scale 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 Node Interaction You may 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 may edit the tree see below The Toolbar The buttons on the toolbar along the top of the viewer allow you to edit the tree If you are viewing a tree made from an alignment the View Seque
136. s in the File menu and in the popup menu when doc ument summary is right clicked Ctrl Click on MacOS 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 9 5 Chat You can either chat with a single contact or invite several contacts to join you in a new chat 9 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 Jabber protocol click on that contact and select New Chat Session either from the Collaboration menu or from the popup menu right click on the contact or Ctrl click on MacOS 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 9 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 con tacts and then select New Chat Session from either the Collaboration menu or the context menu right click on the account or Ctrl click on MacOS Select the online contacts 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 120 CHAPTER 9 COLLABORATION PRO ONLY Accepting or Declini
137. s 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 Also all downloaded files are stored in the destination folder and are marked unread until viewed for the first time 2 6 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 44 CHAPTER 2 RETRIEVING AND STORING DATA 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 this can be clicked to stop the search e Edit Click this to change an agent s database search criteria destination or search in terval e Delete Delete the agent permanently Any documents retrieved by the agent will re main in your local documents 2 7 Filtering and Similar
138. se Alignment section above e 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 e 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 You can also do a clustal alignment via translation and back as with pairwise alignment 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 3 5 SEOUENCE ALIGNMENTS 79 3 5 4 Seguence alignment using MUSCLE Pro only MUSCLE is public domain multiple alignment software for protein and nucleotide seguences MUSCLE stands for multiple seguence comparison by log expectation See http www drive5 com muscle To perform an alignment using MUSCLE select the sequences or alignment you wish to align and select the Alignment button from the Toolbar At the top of the alignment options win dow there are buttons allowing you to select the type of alignment you wish to do Choose MUSCLE here 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 ww
139. se it will wait until its next scheduled search CHAPTER 2 RETRIEVING AND STORING DATA ee Create Agent Search Database PubMed x Match all of the following Deliver To Local 32 9 O New Folder Search every 1 Days C Only get documents created after today Cancel Create and Run Figure 2 9 The Create Agent Dialog 2 6 AGENTS 43 2 6 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 1 Details of the database accessed For example Nucleotide and Genome under NCBI 2 The search type the Agent performed i e 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 following e Next search in x time eg 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 i
140. ser to enter in the database details 11 2 1 Supplying your own Database Driver Server 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 guarnteed that Server 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 11 3 REMOVING A SERVER DATABASE 131 11 3 Removing a server database To remove a server database simply right click on its top folder and choose Remove database 11 4 Administration The typical user will not have to do any administration this section is for those in charge of the database 11 4 1 Groups and Roles Server databases support user groups and roles for managing access to documents This means that you can restrict access of folders to privelleged 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
141. shallow time PMID 16439664 Figure 3 8 Viewing bibliographic information in Geneious BibTex format in a separate tab of the Document Viewer This 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 compatible format This is usually done when creating a bibliography for Microsoft Word documents 3 3 Sequence data Basic technigues such as dotplots and pairwise alignments can be used to study the relation ships between two seguences However as the number of seguences increases methods for determining the evolutionary relationships between them become more complicated When analyzing more than two seguences there are some common steps to determine the ancestral relationships between them The following sections outline the basic tools for prelim inary seguence analysis dot plots seguence alignment and phylogenetic tree building 3 4 Dotplots A dotplot compares two seguences against each other and helps identify similar regions 14 Using this tool it can be determined whether a similarity between the two seguences is global present from start to end or local present in patches The dotplot uses a window of comparison to determine the level of similarity between every pair of sub seguences of length window size in the two seguences being compared 3 4 DOTPLOTS 71 The dots in a dotplot are determined by two
142. sired primers as annotations Then by holding down shift or ctrl command on MacOS also select all of the sequences to test the primers against and 3 7 PCR PRIMERS PRO ONLY 89 g Test Primers Test primers and probes on selected sequences Test all combinations of selected primers on selected sequences Region Input Options 37 _ Included Region Target Region Product Size Between 1003 And 180 C Optimal Product Size 2 More Options Figure 3 15 The primer test dialog choose the same Primers action from the menu and go to Test Primers in the popup menu that appears There are two ways in which Geneious can test your selection of primers and probes The first option in the dialog tests the chosen set of primers and probes on all selected seguences The check boxes beside each primer and probe can be used to specify if it is being tested All selected primer sized sequences and primer annotations can be found in the drop down boxes The second option tests all combination of selected primer seguences as primer pairs on each selected seguence Primer annotations are however omitted from this process To avoid confusion when testing all selected primers annotations will be labeled with the name of the seguence the primer comes from rather than the usual label Warning Using this option can cause the primer test process to take a considerable amount of time to
143. specifying a location Geneious will then present you with options includ ing details on how to download ClustalW and will offer to automatically search for ClustalW on your hard drive To perform an alignment using ClustalW select the sequences or alignment you wish to align and select the Alignment button from the Toolbar At the top of the alignment options win dow there are buttons allowing you to select the type of alignment you wish to do Choose ClustalW here and the options available for a ClustalW alignment will be displayed The options are e ClustalW Location This should be set to the location of the ClustalW program on your computer Enter the path to it in the text field or click the Browse button to browse for the location If the location is invalid and you attempt to perform an alignment Geneious will tell you and offer the options detailed above for getting or finding ClustalW 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 cost Enter the desired gap costs for the alignment e Free end gaps Select this option to avoid penalizing gaps at either end of the alignment See details in the Pairwi
144. st sequence positions Base calling software detects peaks in the four traces and assigns the most probable base at more or less even intervals Vector NTI formats Geneious supports 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 NTI s alignment module 2 2 IMPORTING AND EXPORTING DATA 29 e ma4 pa4 oa4 ea4 and ca6 formats These are the archive formats which Vector NTI uses to export whole databases 2 2 3 Where does my imported data go The above formats can be all imported into Geneious from local files Geneious also enables you to download certain types of documents directly from public databases such as NCBI and EMBL The method used to retrieve a particular piece of data will determine where in Geneious it is stored Data imported from local files This is 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 Data from an NCBI EMBL Contacts search Data downloaded from public databases within Geneious will appear in the Document Table when that database is selected and can be dragged f
145. t be able to tell whether your new contact is online until they accept you as a contact Similarly you will 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 offline 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 whill have a speech bubble behind them 118 CHAPTER 9 COLLABORATION PRO ONLY 9 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 9 5 Rename Contact dialog box 9 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 MacOS the contact in the Services panel and selecting Remove Contact 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 9 3 Sharing Documents Select one of your local folders Select Share Folder from the File menu Alternatively right click Ctrl click on MacOS on a local fold
146. t the primer pair and remove the option of a target region and the ability to use a probe 3 7 PCR PRIMERS PRO ONLY 91 Picking Parameters The advanced options section has two tabs which are available 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 Probe is available if DNA Probe is being designed or tested These two sections are guite similar the DNA probe section has a subset of the options available 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 Most of the options are used 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 During testing many of the absolute limit options are disabled however optimal values can still be set C Maximum ambiguities C Maximum mismatches C Inverse PCR Primer Size Min 18 25 Optimal 204 Max 27 ja Tm Min 57 5 Optimal 605 Max 632 YGC Min 20 2 Optimal 504 Max 80 Product Tm Min 0 2 Optimal 0 5 Max 0 ja Max Tm Difference 100 GC Clamp Of Max Hairpin Score g ja Max Primer Dimer Score Max Poly X 5 j Max 3 Stability g ja Primer Picking Weights a Fewer O
147. ter comparing all the trees in the original set see Consensus Tree section above Topology Threshold The percentage of topologies in the original trees which must be rep resented 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 egual to the number of samples Creating a consensus tree of existing trees If you select a tree set document and choose Tree then the Consensus option will be available at the to of the tree builder options This will create a consensus tree using the trees already in the document no resampling will be performed and it will be added to the tree document A new document will not be generated The only option available here is the consensus support threshold 3 7 PCR Primers Pro only Geneious provides three operations that work with PCR Primers and DNA or hybridisation probes PCR Primers and DNA or hybridisation probes can be designed for or tested on exist ing nucleotide sequences Additionally Geneious can determine the primer characteristics of existing primer sized sequences 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 MacOS on the document s and select Primers A popup menu will appear showing the oper
148. tions BM 2 binii A een Be os ili IN Compress annotations 80 90 100 110 120 130 140 7 549172 YKRRWGAPQR ECAETVATMO AQEANALLES RMEALEWFKK FTWWIRMYAT FIFOHAFSFG EGSMFWEGFP E site 3 Transmembrane protein WES so LI US20 gene C source 1 MIN AAA Hydropho Ih Ran Bu io 2 a v 10 YI gene 1 150 160 70 Tao 190 Ti 549172 QNRNFCWENY SFFETVEVPI MCMFITYTEG NEHPSNATVE FIYHEANSET AALFOMCSES RVEVGSYWMT S protein 1 Hydropho Trans Hide AD gene i Transmembrane region Transmembrane region TS Pana layout Hydropho fit i Ma ae fe ia 220 230 240 250 60 residue 1 gt 342 olors gene US20 product Transmembrane protein HWLF3Foom Options Statistics 342 549172 BABFISFTGH AFEGGRDRRR WKCISCWYNM MEISFETEAN ESDADWEQKE WWT US20 gene o Hydropho Mill bf GH mitt ER 50 1 549172 EMMEFFCPPN OCIRHAWCEY LIMESGPRWE SSDGYPEDN GTETAASTTG KS 488 gt Hydropho iik ae A e AN ee mo Cursor before residue 0 Figure 3 3 The annotations options in the sequence viewer Mouse dragging Click and hold down the left mouse button at the start position and drag to the end position Select from annotations When annotations are available click on any annotation to select the annotated residues Click on sequence name This will select the whole sequence Select all Use the keyboard shortcut Ctrl A to select everything in the panel The availa
149. two tabs that are common to most types of documents Text view and Notes 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 Document button or double click to view it Some document types such as seguences trees and structures have an options panel occupying the right of the document viewer The options in the options panel have an arrow which can be used to expand or hide a group of related options See the next section on document viewers for more information about operating the various viewers in Geneious Most viewers have their own small toolbar at the top of the document viewer panel This always has three buttons on the far right e Expand Document View which expands the viewer panel out to fill the entire main window Clicking again will return the viewer to normal size e Open Document in New Window will open a new view of the selected document in a new separate window 2 1 THEMAIN WINDOW 19 e Help opens the Help Panel and displays some short help for the current viewer 2 1 4 The Help Panel The Help Panel has a Help tab and a Tutorial tab The Help tab provides you 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
150. uction to Geneious its features and how to use them is to watch our online video demonstration http www geneious com demonstration 1 11 Downloading amp Installing Geneious Geneious is free to download from http www geneious com download This down load includes both Geneious Basic free for academic use and Geneious Pro If you are using Geneious for the first time you will be offered a free trial of the Pro features If you have already purchased a license you can enter it when Geneious starts up to activate the Pro features To download Geneious click on the internet address above or type it in to your internet browser to open the Geneious download page then choose your operating system and click Download Geneious Geneious is avaiable for Windows Mac OS X 10 4 Linux and Solaris Once Geneious has downloaded double left click on the Geneious icon to start installing the program 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 then you will only have to double click on the disk image that is downloaded then drag the Geneious application to your Applications folder 1 1 1 Choosing where to store your data When Geneious first starts up you will be asked to choose a location where Geneious will store all of your data The default is normally fine but you may like to store your data on a network
151. ues The ends are trimmed based on these guality values using the modified Mott algorithm Richard Mott personal communication e Trim using ambiguous bases finds the longest region in the seguence with no more N s than the maximum ambiguous bases value and trims what is not in this region This should be used when seguences have no guality information attached e Trim using vector screening against UniVec If your sequences have vector contamination at the ends this option will identify and trim them 3 84 Viewing Contigs Contigs in Geneoius are viewed and edited in exactly the same way as alignments There are several features in the seguence viewer which are worth taking special note of when viewing contigs 96 CHAPTER 3 ANALYSING DATA The consensus sequence is normally of particular interset and this is always displayed at the top of the sequence view labeled Consensus 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 There is a Quality color scheme which is selected by default for alignments of all chro matograms 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 w
152. ur service tree These BLAST programs can be used in exactly the same way as the NCBI BLAST ones 102 CHAPTER 4 CUSTOM BLAST PRO ONLY Chapter 5 COGs BLAST Pro only COGs BLAST allows you to BLAST against the COGs database http www ncbi nlm nih gov C0G Geneious will BLAST your sequence against the COGs database identify which COG the seguence is most likely to reside in and give you information about the COG 5 1 Setting Up To set up the COGs database you first need to set up Custom BLAST on your computer see the section on Custom BLAST Once you have set up Custom BLAST you need to set up the COGs database files 5 1 1 Downloading the COGs BLAST files yourself If you want you can download or otherwise acguire the COGs BLAST database files outside of Geneious You can download them from here ftp ftp ncbi nih gov pub COG COG The files you need are e myva e myva gb e whog Save these files to a local folder Now click on Custom BLAST in the services tree and then click on Add Database Navigate to the file myva and click OK make sure that the protein 103 104 CHAPTER 5 COGS BLAST PRO ONLY database option is checked Now copy the other two files that you downloaded into the data folder inside your Custom BLAST folder 5 1 2 Downloading the COGs BLAST databases through Geneious Geneious provides a download manager to help you download and set up the COGs BLAST database The COGs BLAS
153. urrences of this domain in UniProt seguences for each domain 105 106 CHAPTER 6 PFAM PRO ONLY 3 Pfam B 59 MB contains records for the automatically generated domains in Pfam B taken from PRODOM 4 Pfam C 69 KB contains records for Pfam clans families of similar domains 5 swisspfam 132 MB contains data on the domain architecture of UniProt seguences 6 1 1 Downloading the Pfam databases yourself If you want you can download or otherwise acquire the Pfam databases outside of Geneious You will need to let Geneious know where to look for the files once you have done this To do this select the Pfam service Click the Change Database Location and browse to the location of the databases 6 1 2 Downloading the Pfam databases through Geneious Geneious provides a download manager to help you download the Pfam files To use it select the Pfam Service Click the Let Geneious do it button Then click the Start button After a few seconds the first database will start downloading You can click Pause to pause the download You can search a database as soon as it has finished downloading and its contents have been verified If you shut down Geneious with a file partially downloaded you will need to start downloading it again from the beginning The Pfam databases total around 4 GB in size most of which comes from Pfam A full If your internet connection is slow or you have a low data cap you may want to
154. utorial zip Be careful not to put subfolders in your zip file as these are not supported 8 2 Answering a tutorial Import the tutorial document into Geneious use File Import From file The tu torial 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 accidentaly 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 Docuemnts 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 9 Collaboration Pro only Collaboration allows Geneious pro 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 s
155. ve the currently selected enzymes into a separate document by clicking Save Selected Enzymes The document will be created in the current folder in your local database and this set will then beavailable in the Candidate Enzymes option in this and all future analyses until the document is deleted You can choose a custom name for the document such as Lab Fridge or Enzymes in pBlueScript 11 SK multiple cloning site After configuring your options click OK to start the analysis and annotate the restriction sites on the seguence or Cancel to abort 10 2 Digestinto fragments The option Digest into fragments from the Tools Cloning menu or the context menu allows you to generate the nucleotide seguences 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 e If you have selected only one nucleotide sequence document and it has annotated re striction sites you can select Digest using Annotated cut positions to cut the document on these sites When this option is selected the options to filter the enzymes by their effec tive recognition sequence length or number of hits are disabled However if you select a subset of the enzymes under More Options only the cut sites from these enzymes will be considered this can easily be used for the same effect by sorting by columns and then selecting a range of r
156. view options for any selected service with the right mouse button or by clicking the Options button at the bottom of the Service Panel in Mac OS X 2 12 The Documents Table The Document Table displays your search results or your stored documents While search results usually contain documents of a single type a local folder may contain any mixture of documents whether they are sequences publications or other types If you cannot see all of the columns in the document table you may want to close the help panel to make more room This information is presented in table form Figure 2 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 eg Selecting two sequences will show both of them side by side in the sequence view There are several ways to select multiple documents e Hold Ctrl Command apple key on Mac OS and click to add the document to the current selection e Hold Shift and click to add the document and all documents between it and your previous selection e On windows the right mouse button can be clicked and held while moving the mouse to easily select a block of documents The popup menu will appear once the mouse is released so the newly selected documents can quickly be manipulated 2 1 THEMAIN WINDOW 17 Name Summary VT eat e n Science Roman Biek 16439664 htt
157. w drive5 com muscle muscle html 3 5 5 Combining alignments and adding sequences to alignments Two alignment methods are available in Geneious which allow you to align two alignments together and create a single alignment and align a new sequence in to an existing alignment These are Profile Alignment and Consensus Alignment To perform either of these select the sequences or alignment you wish to align and select the Alignment button from the Toolbar At the top of the alignment options window there are buttons allowing you to select the type of alignment you wish to do Choose Profile Align or Consensus Align here and the options available for your chosen alignment will be displayed Profile Alignment is performed by creating a profile of each alignment sequence and then aligning those using the Needleman Wunsch 16 global pairwise alignment algorithm Profiles are described in the multiple alignment section 3 5 2 Consensus Alignment operates in a similar way to profile alignment except it generates a consensus sequence for each alignment instead of a profile Consensus alignment allows you to choose which align ment algorithm to use for aligning the consensus sequences All of the pairwise and multiple alignment algorithms are available The consensus sequence used for each alignment is a 100 consensus with gaps ignored 80 CHAPTER3 ANALYSING DATA 3 6 Building Phylogenetic trees Genei
158. xt format Newick There are a number of options for the tree viewer Current Tree If you are viewing a tree set this option will be dispalyed Select the tree you want to view from the list General General has 3 buttons showing the different possible tree views rooted circular and un rooted The Zoom slider controls the zoom level of the tree while the Expansion slider expands the tree vertically in the rooted layout Info For a consensus tree the info box displays the consensus method used to build the tree For a topology it also shows what percentage of the original trees have the topology of the displayed tree 66 CHAPTER3 ANALYSING DATA P dl 9 653E 2 EN v General arara Zoom I Expansion I 8 037E 2 v Layout 03762 visa Root Length J Curvature J J Align Taxon Labels v Formatting 7 Transform branches C Order branches 0 182 OChicken 0 03 Iv Show Root Branch a NN Bi Lino AMoiohks 1 1 Figure 3 6 A view of a phylogenetic tree in Geneious 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 views 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 t
159. xy server Please see step 3 1 3 TROUBLESHOOTING 13 e Use auto config file This enables one text field called Config file location These details can also be found in your browser s settings 6 Set the proxy host and port settings under the General tab to match those in your browser 7 If your proxy server reguires 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 email us at support geneious com A e Tools Preferences Tools Internet Advanced Options Options Proxies Connection Connections Settings Connection Lan Settings Settings Figure 1 5 Checking browser settings 1 3 3 Weblinksinside Geneious don t work under Linux Set your BROWSER environment variable to the name of your browser The details depend on your browser and type of shell For example if you are using Mozilla and bash then put export BROWSER mozilla in your bashrc file When using a csh shell variant put setenv BROWSER mozilla in your 7 cshrc file 14 CHAPTER 1 GETTING STARTED 000 Preferences General Plugins General settings Data Storage Location Users alexei Geneious Search history M Check for new versions of Geneious Also check for beta versions of Geneious Use browser connection settings eH Proxy host Connection setting
160. y once or twice as well as a region enzymes may not cut within After running the analysis the position of the matching enzymes recognition sequence and the sites where they cut will be visible on the sequence as annotations and you will be able to see a table of all fragment start and end positions and their lengths and of all restriction enzymes involved These tables can be exported as csv files for subsequent processing with other software such as e g Microsoft Excel Like many restriction enzymes EcoRI is methylation dependent and cuts only if the second A in the recognition seguence is not methylated to N6 methyladenosine 2The restriction enzyme information included in Geneious was obtained from Rebase 18 available for free at http rebase neb com 121 122 CHAPTER 10 CLONING PRO ONLY e Digest into fragments allows you to generate the actual fragments that would be created in a digestion experiment using restriction enzymes When running a digestion experi ment you can choose to either use the restriction sites already annotated to the seguences or a subset that corresponds to only some specific enzymes or you can let Geneious de termine the cut sites for any candidate enzymes The latter option finds the cut sites for the candidate enzymes and generates the fragments in a single step e Ligate Sequences lets you ligate two or more fragments with or without overhangs e Insert into Vector allows you to

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