Basic question about local BLASTP

[Tsuyoshi]

Hello.

I am using local BLASTP program to blast my protein sequences (around 600 sequences) against other organism's whole proteome database. What I want to do is to filter the output by some parameters. I have applied a reciprocal best_hit strategy, and the parameters were set as 'max identity > 30%', e<1e-10 and best_hit_overhang 0.25). However, in some cases, for example, the max identity between one best-hit set of query and subject sequence was as high as 40%, the query coverage was only 2%. Therefor, I want to exclude the best-hit sequences like that by setting a parameter like 'query coverage > 30%'.

The web based blast on NCBI could show the query coverage, but I do not know what is the command line for local BLASTP to output "query coverage". Please give me some ideas to resolve this problem. Thanks!

[mknut]

You can try this:

Update to the newest BLAST+ package.
Use

-outfmt

, which allows the output to be in custom format. You can find documentation on custom output format here (BLAST Command Line Applications User Manual, Appendix C, option 'outfmt'). From what I see the values to use are

-outfmt 6 qcovs

, which should add query coverage per sequence value at the end of each output line and save it in tab-delimited format.

[Tsuyoshi]

You can try this:

Update to the newest BLAST+ package.
Use

-outfmt

, which allows the output to be in custom format. You can find documentation on custom output format here (BLAST Command Line Applications User Manual, Appendix C, option 'outfmt'). From what I see the values to use are

-outfmt 6 qcovs

, which should add query coverage per sequence value at the end of each output line and save it in tab-delimited format.

Thank you mknut.

I tried adding qcovs, however, the output files did not contain the qcovs information. For example, if I input the command as outfmt "qacc sacc evalue bitscore qcovs", the output files only contained qacc sacc evalue bitoscore. I am thinking whether qcovs is only available for blast but not for blastp.

[GenoMax]

I am thinking whether qcovs is only available for blast but not for blastp.

The manual seems to indicate that those options are available for all blast+ programs. Have you tried 'std qcovs'?

Or perhaps the command needs to be (note: single quotes):

-outfmt 6 [COLOR="Red"]'[/COLOR]qcovs[COLOR="Red"]'[/COLOR]

[mknut]

Table C1: Options common to all BLAST+ search applications.

So yes, it seems that this command should be applicable to BLASTp. It also seems that the proper syntax to use is with double quotes, containing numerical argument as well as the rest (single quotes might work) so:

-outfmt "6 qcovs"

If this does not work, you might want to think about calculating the coverage % with a script, taking query lenght from your BLASTp query and alignment length from 'length' of BLASTp output.