Frequently Asked Questions (FAQs)

Q: I cannot connect to the database (message: Could not connect to database: publicdb.blast2go.com)

A: Check the following:

• The mySQL port (3306) is open at your institution for out-going connections (ask your sys-admin)
• You do not have any personal firewalls installed (in Windows under System Preferences)
• Do you have the right database name (host-name or ip-address) and DB-Name (see Launch B2G) 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:

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 Fisher's Exact Test implementation 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: 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 Fisher's Exact Test implementation 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.

Q: What is the BDA (Blast2GO Descriptor Annotator)?

A: The BDA, in a first step, filters out words from an exclusion list, ID and ACC as well as character and number codes. In a second step, it counts the frequency of word for all blast hit descriptions and transfers the most frequent ones to the query sequence.Users can de-activate or undo the BDA for some sequences results going to Menu→Tools→Reset BDA, or change the description of sequences manually, under context menu.

Q: I am working in Windows and trying to install the B2G 2048 version and I get the error message: “Could not create the JAVA Virtual Machine”. What is the problem?

A: The maximum amount of memory which can be used by a Java Application like Blast2GO depends also on the system architecture, operation system (OS) and the Java version you are using. To go beyond the 1800MB (Linux and Mac) or 1500MB (Windows) of memory you have to use a 64bit OS and a 64bit JAVA version. These which can only be run on a 64bit computer system (hardware).

Q: Can I blast against a different database than those provided by default in Blast2GO?

A: In case you want to blast against a different database you have two options:
If the database is at the NBCI (you can check at http://www.ncbi.nlm.nih.gov/staff/tao/URLAPI/remote_blastdblist.html) you just have to add the name of this database at the Blast.blastdbs line of the blast2go.properties file and use this db instead of nr for remote blast. For example, if you want to blast specifically against “arabidopsis genbank protein” you have to add at the blast2go.properties file the string “gp/3702.9506/at_genbank_prot” , like this:

Blast.blastdbs=nr,swissprot,gp/3702.9506/at_genbank_prot

Open Blast2GO again and this db will be available for selection at the Blast Configuration menu.

If your specific database is NOT at this server, but somewhere else, then you have to provide at the blast.properties file the url of the blast server, adding this at the Blast.urls line, and also the name of the database at the Blast.blastdbs. When you open B2G, you can select then the new blast server url and the blast db. You also have to change then Blast Mode to WWW-Blast at the Blast2GO Blast Configuration Menu.
Please, note that blasting against an EST or other nucleotide database will allow you to recover blast results but NOT to annotate you sequences with GO terms. For GO term annotation you NEED to blast against a protein database since GO terms are assigned to proteins.