Q: I cannot connect to the database (message: Could not connect to database at: 193.144.127.204)
A: Check the following:

• The mySQL port (3306) is open at your institution
• You do not have any personal firewalls installed
• You have the right IP address and DBname (see B2G homepage) selected (in the B2G menu under Tools –> DB Configuration)

Q: I do get blast hits but the mapping step returns no GOs
A: This is most probably due to a wrong blast program. Check you run blastx (you need to get protein IDs, since GOs are assigned to proteins)

Q: Some of my sequences have low blast e-values and mapping results but failed to get annotated. Why?
A: B2G annotation algorithm uses similarity values and evidence code weights (ECw)for annotating. Default values were chosen to provide a good balance between quantity and quality of annotation. Sequenced that failed to be annotated despites a low e-value in the Blast result and a positive mapping could be because: i) these low e-values are only for blast hits without mapping data ii) despites low e-values, similarity values are still low, iii) Evidence Codes are of low reliability and these mapping data is excluded at annotation as these EC has low weights assigned. If users can still create an anntoation result from their data, annotation parameters can be changed: either lowering annotation cut-off or rising ECw (under annotation menu). In this case it is recommended to check a few sequences manually to see how annotation results changed after modifying parameters.

Q. The blast does not work. I get this error message:

Undefined error when performing BLAST at NCBI for null. Please check parameters and input data (blastx, blastp, etc.)
12:51 - Used NCBI info request was: http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?CMD=Get&TOOL=blast2go&EMAIL=your@mail.here&FORMAT_OBJECT=SearchInfo&RID=null

A: Check you do not have strange symbols in the sequence names such as ”#” or ”?”, etc.

Q: In the enrichment analysis, why aren't the outputs if you switch the test and reference sets the same but in opposite direction? A: This is due to how the Gossip package (which implements enrichment analysis in Blast2GO) works. Here, the genes in test set that are present in the reference set are removed from the reference but not from the test. This is needed to create a consistent contingency table used in the Fisher's Exact Test. For most applications of enrichment analysis in microarray data, this is what you should do. For other comparisons (i.e. comparing 2 libraries), then duplicates genes would need to be removed from both lists. This option is available at the Enrichment Analysis menu.

Q: Blast2GO does not open/show Blast-Result links in a web browser, how to fix this?
A: Open the “blast2go.properties” file in the directory “blast2go” in you home directory and change the settings for “BlastBrowser.Explorer”:

• Browser settings:
  • Linux:
    • BlastBrowser.Explorer=/usr/bin/firefox
    • BlastBrowser.Explorer=/usr/bin/konqueror
  • Windows:
    • BlastBrowser.Explorer=C:\Archivos de programa\Internet Explorer\iexplore.exe
    • BlastBrowser.Explorer=C:\Archivos de programa\Mozilla Firefox\firefox.exe
  • Mac:
    • BlastBrowser.Explorer=/usr/bin/open

Q: How to preform a Fisher Exact Test when I want to compare 2 group of sequences which I have in different annotation files?
A: For performing the Fisher test what you have to do FIRST is to have ALL your sequences (from Group1 and Group2) loaded in the application, and then select test and reference groups.
Thus basically:

• Join your Group1.annot and Group2.annot file in one annotation file, All.annot
• Upload All.annot in blast2go (From the File menu)
• Create 2 lists of sequences (only sequence IDs), one with the Group1 sequences and other with the Group2 sequences. Give these files the extension .txt
• Go to the Fisher Exact test
• Select Group1.txt as test group
• Select Group2.txt as reference group

Remember that the Gossip implementation of the Fisher exact test is sensitive to the direction of the test: the sequences that are in Group1 and also in Group2 are removed from Group1, but not from Group2. If you have sequences in common in both lists and want to perform a test which is insensitive to the direction of the comparison, select the option “remove duplicates” when performing the test.

Q: For some of the sequences I am failing to obtain a blast result using default parameters. They remain white. Why?
A: Failing a blast result to come back from the NCBI server is out of Blast2GO hands. The reason is connectivity limitations between you and the NCBI server. Here the only thing to do is improve connectivity at your site or re-run the blast.

Q: How do I obtain all the sequences present in a given slide (GO term) of the pie charts or node of the Combined Graph?
A: When you create the combined graph (prior to the pie charts), there is an option to export the graph information as txt file. This is represented by an icon with the letters txt on the graph top bar. In this file you can find all sequences annotated to each term present in the combined graph or pie chart.