This section provides a quick run-through of a basic functional annotation process done within Blast2GO. More detailed descriptions of the different analysis steps and more advanced features are described in the remaining sections of this documentation.
Go to File Load > Load Sequences > Load Fasta File and select your .fasta file containing the set of sequences in FASTA format. Alternatively you can load the example sequences into Blast2GO choosing File > Load > Load Example Sequences. Please download example files to try and test Blast2GO: b2g_example_files.zip
Click on the orange blast toolbar icon. In the BLAST Configuration Dialog (Figure 1) select the type of BLAST mode which is appropriate for your sequence type (blastx for nucleotide and blastp for protein data), click Next for the advanced settings and to choose where to save the Blast results and click Run to start the BLAST search against NCBI’s non redundant NR database. Alternatively you can choose to use different BLAST such as CloudBlast, AWS Blast and Local Blast on the arrow next to the blast (toolbar). See BLAST Section for further details on this.
- Once your BLAST analysis is finished visualize your results at charts (toolbar) > BLAST Statistics.
On the Main Sequence Table, right-click on a sequence to open the Single Sequence Menu (Figure 7). Select Show BLAST Result to the BLAST Browser for that sequence.
If you are running blast using CloudBlast we recommend to run blastx-fast or blastp-fast as it is faster and less computation units will be consumed.
By clicking on the interpro icon the corresponding Wizard will be shown. Please provide a valid email address. Since this search is executed via a web service provided by the EBI. It is highly recommended to run IPS in order to improve the quality of the annotations. Once InterProScan
Results are retrieved use Merge InterProScan GOs to Annotation to add GO terms obtained through motifs/domains to the current annotations. InterProScan can be run in parallel with BLAST.
Click on Mapping icon > Run Mapping to start mapping GO terms. Mapped sequences will turn green. Once Mapping is completed visualize your results at charts (toolbar) > Make Statistics > Mapping Statistics.
Click on annot (toolbar) to open the Annotation Configuration Window. Click Next to change the evidence codes and finally click Run to start the annotation. Annotated sequences will turn blue.
- Once the annotation is completed you are able to visualize your results with charts (toolbar) > Statistics Annotation Statistics.
- On the Main Sequence Table, right-click on a sequence to open the Single Sequence Menu. Select Draw Graph of GO-Mapping with Annotation Score to visualize the annotation on the GO DAG for that sequence.
- If desired, modify the annotation by clicking with the right mouse button and select Change Annotation and Description or change the extent of annotation by adding implicit terms Annotation (arrow) > Run ANNEX or reducing to a GO-Slim representation Analysis > GO-Slim > Run GO- Slim (online).
- During the annotation process, Enzyme Codes (EC) will be also given when a GO-term/EC number equivalence is available.
Blast2GO provides tools for the statistical Analysis of GO term frequency differences between two sets of sequences. Go to Analysis > Enrichment Analysis (Fisher's Exact Test) and a new Dialog window will open (Figure 1). Select a .txt file or an ID list containing the sequence IDs for a subset of sequences. A test-set example file can be downloaded the Blast2GO website. Select a second set of sequences as reference/ set if desired. If no reference set is provided all annotations of the corresponding project will be used as reference. Click Run to start the analysis. A table containing the results this analysis will be displayed in a new tab.
- Click on Make Enriched Graph icon to visualize the results of the Fisher’s Test on the GO DAG.
- Click on Show Bar Chart to obtain a bar chart representation of GO frequencies.
- The results can be reduced to more specific GO terms in the corresponding icon and saved as text format (Save as Text).
- Combined Graph
Blast2GO can visualize the combined annotation for a group of sequences on the GO DAG. Select a group of sequences to generate their combined graph at select (toolbar) > Select Sequence. Now Select by Features and Select by Name or ID. You can use the Demo Test Set used previously for this. Alternatively, you can select sequences using the sequence check boxes of the Main Sequence Table. Now go to graphs (toolbar) > Make Combined Graph. Now click Run.
- Save Results
File > Save saves the current Blast2GO project as .b2g file.
- Export Results
- File > Export allows to export the generated data in many different formats.
- File > Export Annotations exports the actual annotation results as .annot file or generate own formatted annotation file as .txt file.
- The enrichment analysis results can be exported in various formats from the Fisher Exact Test Result Viewer. ‘‘Save as Text’’ exports the results as a tabulator separated text file.
- To export GO graphs use the sidebar of the corresponding graph viewer. Graphs can be saved/exported in .pdf, .png, .svg and .txt.