blastn command line returning empty file

Question

[gimenezenrico]

Hi there!

I'm trying to automate some blast searches with biopyhton, but instead of returning me the .txt file, it creates an empty file. I've tried to run it direct in linux terminal, but the result was the same.

in biopython:

from Bio.Blast.Applications import *

path = str(input("Type the path to fasta file: "))

comando_blastn = NcbiblastnCommandline(query= path, task="blastn-short", remote=True, db="nt", \
outfmt='6 qseqid qcovs sscinames pident evalue', out= "out.txt")
print(comando_blastn)

stdout, stderr = comando_blastn()

blast_result = open("out.txt", "r")

lines = blast_result.read()
print(lines)

in terminal:

blastn -out outComandLine.txt -outfmt "6 qseqid qcovs sscinames pident evalue" -query Ensaio_toxo.fasta -db nt -remote -task blastn-short

I'm using -task blastn-short due to length of my sequences, but I've already tried without it.

I'm quite new to this, so I wouldn't be surprised if there's some mistake in my code.

Does some one know what could be going wrong here?

EDIT:

I tried to see if there was any hits, so I ran:

blastn -query Ensaio_toxo.fasta -db nt -remote -task blastn-short -out outComandLine.txt

And the result was the following:

BLASTN 2.12.0+

Reference: Stephen F. Altschul, Thomas L. Madden, Alejandro A.
Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J.
Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs", Nucleic Acids Res. 25:3389-3402.


Database: Nucleotide collection (nt)
           81,664,709 sequences; 723,646,484,289 total letters

Query= FwToxo
Length=18
RID: 6N1SGV3V013

***** No hits found *****

Lambda      K        H
    1.37    0.711     1.31 

Gapped
Lambda      K        H
    1.37    0.711     1.31 

Effective search space used: 1444679697890

  Database: Nucleotide collection (nt)
    Posted date:  Apr 23, 2022 07:59 AM
  Number of letters in database: 723,646,484,289
  Number of sequences in database:  81,664,709

Matrix: blastn matrix 1 -3
Gap Penalties: Existence: 5, Extension: 2

In command line it returns no hits, but when I run it on Web BLASTn platform it returns hits normally. It seems that there is some problem with BLAST command line, not exactly with the code...

[GenoMax]

I've tried to run it direct in linux terminal, but the result was the same

That would indicate that there is a problem with your command line. Can you show us the command line you used for the direct search (not via biopython).

[gimenezenrico]

I used this:

blastn -out outComandLine.txt -outfmt "6 qseqid qcovs sscinames pident evalue" -query Ensaio_toxo.fasta -db nt -remote -task blastn-short

and runed on terminal.

[Ram]

Add a 2> cmd.err to your command and show us the contents of that file once you run it.

[gimenezenrico]

The result was the following:

Warning: [blastn] Taxonomy name lookup from taxid requires installation of taxdb database with ftp://ftp.ncbi.nlm.nih.gov/blast/db/taxdb.tar.gz

Then I installed the taxdb, and ran again. The result remains empty... The cmd.err file was empty after that too.

[GenoMax]

There is something wrong with your query file. Can you show us output of grep -A 2 "^>" Ensaio_toxo.fasta? If you take some sequences from that file and run a search directly at NCBI using the web interface do you get results?

Since you are running the search remotely at NCBI there is no point in installing the taxonomy database locally since it will not be used.

[gimenezenrico]

grep -A 2 "^>" Ensaio_toxo.fasta returns my sequences inside the file normally. When I run it directly at NCBI web interface it returns me results normally. The problem is just with the command line application. If I run without -outfmt 6 it returns me the following:

BLASTN 2.12.0+


Reference: Stephen F. Altschul, Thomas L. Madden, Alejandro A.
Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J.
Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs", Nucleic Acids Res. 25:3389-3402.



Database: Nucleotide collection (nt)
           81,664,709 sequences; 723,646,484,289 total letters



Query= FwToxo

Length=18

RID: 6N1SGV3V013


***** No hits found *****



Lambda      K        H
    1.37    0.711     1.31 

Gapped
Lambda      K        H
    1.37    0.711     1.31 

Effective search space used: 1444679697890


  Database: Nucleotide collection (nt)
    Posted date:  Apr 23, 2022 07:59 AM
  Number of letters in database: 723,646,484,289
  Number of sequences in database:  81,664,709



Matrix: blastn matrix 1 -3
Gap Penalties: Existence: 5, Extension: 2

[GenoMax]

I run it directly at NCBI web interface it returns me results normally.

Then I suggest that you make a note of the parameters (you will find them under Search Summary) that the web application is using and try to replicate them exactly on local command line.

[gimenezenrico]

Thanks GenoMax ! It worked! maybe this -task blastn-short isn't working properly.

I used the following command in shell:

blastn -query Ensaio_toxo.fasta -db nt -remote -out outComandLine.txt -evalue 1000 -word_size 7 -penalty -3 -reward 1 -outfmt "6 qseqid qcovs sscinames pident evalue"

But to use the sscinames option in -outfmt 6 you will have to download taxid database.

[GenoMax]

I assume query file contains one or more fasta formatted sequences? If not that could be one issue. Can you try the variation below?

blastn -query Ensaio_toxo.fasta -db nt -remote -task blastn-short -out outComandLine.txt -outfmt "6 qseqid qcovs sscinames pident evalue"

[gimenezenrico]

Yes, it has pries and probes in fasta format. I tried this variation and the result was the same...an empty file.