Error in run blastpgp

Question

[thanhhieu43]

Hi,

I am trying to have the command line blast work, however, I did not manage to execute it properly. I would appreciate if you can tell me what I am doing wrong.

So here are the things I did so far:

1. I downloaded the Win-64 executable file from NCBI/blast/latest (2.2.26). Double-click and install the files under "C:\blast"
2. I went to Environmental Variables window and created variable BLAST with value "C:\blast\bin", BLASTDB with "C:\blast\db", BLASTMAT with "C:\Blast\data".
3. Under "C:\blast", I have three folders: bin, doc and db folder that I created to put the fasta database.
4. I downloaded pre-formatted non-redundant (nr) fasta database from NCBI ftp and put it under C:\blast\db
5. I used formatdb -i c:\blast\db\nr -p T -o T command to create index files.
6. Now C:\blast\db has the following files: nr.00.phr, nr.00.pin, nr.00.psq, [...], nr.08.phr, nr.08.pin, nr.08.psq an

7. Next I wanted to generate pssm file using this command:

   C:>blastpgp -i c:\blast\O00257.fasta -j 2 -d c:\blast\db\nr -Q O0257.pssm
   

Unfortunately, I get the following error:

[blastall 2.2.24] WARNING: Unable to open nr.00.pin
[blastall 2.2.26] WARNING: Unable to open nr.00.pin
[blastall 2.2.26] WARNING: Unable to open nr.00.pin
[blastall 2.2.26] WARNING: Unable to open nr.01.pin
[blastall 2.2.26] WARNING: Unable to open nr.01.pin
[blastall 2.2.26] WARNING: Unable to open nr.01.pin
[blastall 2.2.26] WARNING: Unable to open nr.02.pin
[blastall 2.2.26] WARNING: Unable to open nr.02.pin
[blastall 2.2.24] WARNING: Unable to open nr.02.pin
............................................................................
[blastall 2.2.26] WARNING: Unable to open nr.08.pin
[blastall 2.2.26] WARNING: Unable to open nr.08.pin
[blastall 2.2.26] WARNING: Unable to open nr.08.pin

My understanding is that it complains that it cannot locate the nr database. However, I already point to the correct directory when I set it in the environmental variables.

What is the problem here?

Please, help me.

My email: [e-mail], [e-mail]

Answer 1

[hpmcwill]

Firstly if you are setting up NCBI BLAST for the first time, it is recommended that you use NCBI BLAST+ instead of the legacy NCBI BLAST.

In your "Command Prompt" check that the environment variables are actually set in the shell environment, for example to check BLASTDB:

echo %BLASTDB%

If this does not return the expected file system path (e.g. C:\blast\db), you can set it directly in the shell:

set BLASTDB=C:\blast\db

To check if the BLAST database(s) can be found try:

fastacmd -d nr -I

for legacy NCBI BLAST, or for NCBI BLAST+:

blastdbcmd -db nr -info

This will report some information about the database, if it is found, or an error message.

If the database is found, then it might be something wrong with the database. Given that you are using the NCBI supplied version of the 'nr' database you may need to re-download the archives, and unpack them (i.e. repeat step 4). Since the database is supplied as a pre-built NCBI BLAST database you should not attempt to reformat it (i.e. skip your step 5).

[thanhhieu43]

I checked all according to your suggestion, all right. But the same error occurs. Finally I downloaded the database from the files nr.##.tar.gz (##: 00 -> 24), then I just unpacked (not execute formatdb) and this time when performing command: blastpgp, this time no have error occurred as before.

I thank you for helping me.

I also tried installing the NCBI Blast +, but I do not know how to create a file PSSM from file fasta. Please guide me the command and syntax command which to do that. Thanks!

Best regards,

Thanh Hieu.