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Q: I cannot connect to the database (message: Could not connect to database at:
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)

Additional tips (kingly provided by Rick Westerman)

“First, when having problems, the advice is to erase the blast2go program and the blast2go properties files from your computer and start fresh. This will solve most problems.

However if you are continuing to have problems then another thing we can do is to make sure that the network port is actually open from *your* computer. Not open as per what someone such as your network person says should be. Not open from looking at someone else's computer. But rather from *your* computer. (Story below). To do this we will use the 'telnet' program in order to side-step any problems that might be due to B2Go. 'Telnet' usually goes to port 23 but most versions of it can also go to other ports and specifically port 3306 which is the one that the B2Go server uses for annotation. Unfortunately telnet does vary a bit from operating system to operating system so you may need to change the instructions below. They did work from WinXP, MacOS 10.4 and Ubuntu 10. The following is from my Mac.

Get to the command line and type in the program, address and the port like so:

telnet 3306

You should see something like:
Connected to
Escape character is '^]'.

The key point to look for is that '5.0.32-Debian' portion of the line. If you get that then you have connected to the B2Go data base. The connection should time out and you'll will get something like:

Connection closed by foreign host.

But that is fine. Just get that 'Debian' response.

If you do not get the response – especially if you do at least two attempts at least 24 hours apart – then you have some sort of network problem. Something somewhere is blocking your network. Solving this may take quite a bit of effort. However getting your network guru involved will help.

If you do get the response then something about your copy of blast2go is the problem. If deleting your copy of B2Go and the B2Go parameters file does not cure the problem then send email to the blast2go group list.”

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:

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 “” 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.

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: You might have an available memory limitation problem. You can reduce the memory requirement dynamically to 1800 or 1700. TO do this, install B2G using the link “” or “”.

Q: Can I blast against a different database to those provided by default by 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 you just have to add the name of this database at the Blast.blastdbs line of the 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 file the string “gp/3702.9506/at_genbank_prot” , like this:


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 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.

faq.txt · Last modified: 2010/08/30 18:30 by aconesa
Bioinformatics and Genomics Department
Centro de Investigación Príncipe Felipe
Valencia, SPAIN
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