Blast2GO PRO Plugin for Geneious User Manual Geneious 8.0 Version 1.0 October 2015 BioBam Bioinformatics S.L. Valencia, Spain Contents Introduction 2 1.1 Blast2GO methodology . .2 1.2 Blast2GO and BioBam . .3 1.3 Geneious and Biomatters . .3 Quick-Start 4 User Interface Overview 6 3.1 Toolbar . .6 3.2 Document Table . .6 3.3 Document Viewer . .7 3.3.1 Statistics Tab . .7 3.3.2 Graph Viewer Tab . .9 3.4 Sources Panel . 11 Blast2GO functions 13 4.1 Load Example Data . 13 4.2 Convert to Blast2GO . 13 4.3 Add Blast Hits . 13 4.4 CloudBlast . 13 4.5 Add InterProScan . 14 4.6 InterProScan . 14 4.7 Mapping . 15 4.8 Show GO Description . 15 4.9 Annotation . 15 4.10 Merge InterProScan . 16 4.11 ANNEX . 16 4.12 GO-Slim . 16 4.13 Remove Results . 18 4.14 Fisher's Exact Test . 18 4.15 Selection . 19 4.16 Activate Subscription Key . 20 4.17 Remove Subscription Key . 20 4.18 CloudBlast History . 20 Geneious functions 21 5.1 Import Data . 21 5.2 Export Data . 21 5.3 Workflows . 21 Copyright 2015 - BioBam Bioinformatics S.L. 1 Introduction Support: [email protected] Website: http://www.blast2go.com/geneious-plugin 1.1 Blast2GO methodology Blast2GO Conesa et al. (2005) is a methodology for the functional annotation and analysis of gene or protein sequences. The method uses local sequence alignments (BLAST) to find similar sequences (potential homologous) for one or several input sequences. The program extracts all GO terms associated to each of the obtained hits and returns an evaluated GO annotation for the query sequence(s). Enzyme codes are obtained by mapping from equivalent GOs while InterPro motifs can directly be queried at the InterProScan web service. A basic annotation process with Blast2GO consists of 3 steps: blasting, mapping and annotation. These steps will be described in this manual including further explanations and information on additional functions G¨otzet al. (2008). Figure 1.1: Table and Graph Viewer visualizing a small data-set Copyright 2015 - BioBam Bioinformatics S.L. 2 1.2 Blast2GO and BioBam Blast2GO PRO Plugin for Geneious is developed, maintained and distributed by Biobam Bioin- formatics S.L. 1.3 Geneious and Biomatters Geneious is a powerful and comprehensive suite of molecular biology software tools. Geneious provides a an easy-to-use interface with simple and intuitive data management capabilities within a customizable and extendable platform. This allows researcher working with sequencing data to gain immediate access to a wide-range of essential data analysis features. Blast2GO PRO is now part of this feature-set in form of the plugin described in this document. Geneious is developed by Biomatters, a New Zealand based company, founded in 2003, with a mission to create bioinformatics solutions for the analysis, interpretation, and application of molecular sequence data. Copyright 2015 - BioBam Bioinformatics S.L. 3 Quick-Start This section provides an overview of an typical Blast2GO usage. Detailed descriptions of the different steps and possibilities of this plugin are given in the remaining sections of this manual. 1. To start an annotation process with Blast2GO load a sequence file in fasta format containing your nucleotide or amino acid sequences: File Import From File . Alternatively you may use any sequence data already available in the Sources navigator on the left. 2. Convert your data to Blast2GO sequences: Select the sequence document list (or one or various loose sequence documents) and Right Click Blast2GO Convert to Blast2GO . This converts your sequences in Blast2GO sequences and activates three Blast2GO viewer in the bottom editor area (B2G Table, B2G Statistics, B2G Graph). You can now select B2G Table to view the sequence names in a list. Ob- serve that all sequence rows are white and to not contain information. Select the B2G Statistics Tab and click on the white colored statistic names in the right side panel (one of the first 4 options. 3. Blast sequences: Various options are available to add sequence alignments to your Blast2GO sequences in Geneious. We will test the CloudBlast option. More information about other options can be found in the section Blast. Right Click Blast2GO CloudBlast In most cases default settings can be applied. However, depending on the data-set the following parameters might be adjusted: • Select a Blast DB that fits your data-set. • In the advanced section (More Options), choose where a copy of your Blast results should be created. Now sequences will be send to the CloudBlast resource. The progress can be observed under the Sources panel. While processing, the color of the B2G Table changes from white to red or orange. Right-clicking on an orange sequence allows to Show Blast Result via a context menu. This option opens an extra window called Blast2GO viewer component. This is an independent window containing a list of result tabs. These tabs will not change its contents even a user changed to a different data-set (as the integrated Document Viewers). Once we have obtained Blast results the corresponding statistics can be viewed in the B2G Statistics viewer. 4. Perform Gene Ontology Mapping: Orange sequences contain at least one blast hit (probably more than one) and are suitable for the mapping (the number of hits is shown in the #hits column in the B2G Ta- ble). Select all blasted sequences (whether they are white, red or orange) and Right Click Blast2GO Mapping . Now orange sequences may changed their color to green. Green se- quences contain GO candidate term which will be considered for annotation in the next step. 5. Annotation: Once GO mapped sequences (green) it is possible to apply the annotation step: Right Click Blast2GO Annotation We will use default parameters. Some sequences will now change from green to blue and are know successfully annotated. We can now use the green and blue options from the B2G Statistics viewer to review the annotation process. Annotated sequences (blue) can also be visualized with the B2G Graph tab. Copyright 2015 - BioBam Bioinformatics S.L. 4 6. InterProScan: To complement the blast-based annotations with domain-based annotations, run an In- terProScan search. Select your data-set, Right Click Blast2GO InterProScan , introduce your email address and press OK. This adds information to the InterPro column in the B2G Table viewer, which must be merged to your already obtained GOs with Right Click Blast2GO Merge InterProScan . 7. Export Results: Once the annotation process is finished we can consider several options to export the results: File Export Selected Documents. The Files of Type combo-box shows various possible ex- port types and the ones beginning with Blast2GO are most suitable for our data-set. • annot-file: The annot file is the standard format to export GO annotations. It is a tab-separated text file, each row contains one GO term. • dat-file: The standard Blast2GO project file. This file can also be opened with the standalone Blast2GO application. Copyright 2015 - BioBam Bioinformatics S.L. 5 User Interface Overview This section provides an overview of the Blast2GO PRO Plugin for Geneious user interface. 3.1 Toolbar By default, the main Geneious toolbar hosts an icon for all Blast2GO functions and a Blast2GO toolbox icon. The functions icon provide access to the algorithms like Blast, Mapping or Anno- tation. The toolbox contains options like Activate Subscription or the CloudBlast History viewer. All menu options are also available via the context menu of the Document Table (via right-click on a document). Figure 3.1: An overview about the graphical elements the Blast2GO plugin makes available to the user 3.2 Document Table The Document Table shows all available documents. Within the plugin we treat sequences documents (nucleotide or amino acid) which have been converted into B2G documents. These sequences can be loose sequences or grouped into lists of B2G Sequences. Note: At the moment Blast2GO list-documents can not be distinguished by their icon from normal nucleotide or amino-acid lists (Figure 3.3). Copyright 2015 - BioBam Bioinformatics S.L. 6 (a) Group sequences into a list document (b) Extract sequences from list Figure 3.2: Managing sequences and lists of sequences Note: The Blast2GO viewers works significantly faster with un-grouped sequences. Un-group sequences requiring more RAM and this is not feasible when working with large lists. 3.3 Document Viewer The Document Viewer in the lower-right area visualizes the data selected in the Document Table. There are 3 different viewers to visualize grouped or un-grouped B2G Sequences: B2G Table, B2G Statistics, B2G Graph • B2G Table Tab The Blast2GO table shows the obtained results for each sequences including the analysis status (blast, mapped, annotated, etc.). When working on a list, selection can also be performed via the section tool (toolbox). • B2G Statistics Tab The statistics viewer can be used to create charts for all analysis steps and allows to export results in various file formats via the sidepanel. Statistics related to blasted sequences are colored in orange, annotation step related statistics are blue etc. Note: A dataset with green sequences e.g. can not be used to generate e.g. blue statistics. A GO Distribution Level statistic for example does not make any sense when applied on only mapped sequences. Section 3.3.1 gives an overview about the different available charts and their meaning. • B2G Graph Tab To visualize a GO combined graph the viewer offers many different options in the sidepanel (Figure 3.3(b)). A first execution had to be started via the Make it so button. Subsequent parameter changes of the Graph Options have a direct impact on the shown graph.