<p>I was running tblastn using standalone blast2.9.0+ through Ubuntu Linux, to get the alignment result of a list of protein sequences for same species, against its complete genome file. </p>

<p>The command I was using: </p>

<p><code>~/softwares/ncbi-blast-2.9.0+/bin/tblastn -query TBLASTNstring/882.Tstring -subject ../com_gen/*882*.fasta -outfmt "7 acc qacc pident qcovs evalue sstart send sframe" -max_hsps 1 -num_alignments 1 -out 882.blastout</code></p>

<p>(Info update: The query file has more than 1 sequence but all are in fasta format; the subject file has only 1 sequence; there is only 1 file with "882" named in the folder)</p>

<p>And I received error from tblastn as follow:</p>

<pre><code>Error:  (CArgException::eSynopsis) Too many positional arguments (1), the offending value: ../com_gen/DP_882_DeVul_Hilde.fasta
</code></pre>

<p>All the sequences in both my query and subject sequence files are of two lines, the header line started with ">", followed with sequences.</p>

<p>I have checked both query and subject sequence files using <code>sed -n '0~2p' | grep -v -E '[[:alpha:]]'</code> to see if there exist special character or space within the sequence line, but the output of the results return nothing. </p>

<p>PS: the complete genome file contained of nucleotide sequences with only ATGC (I have checked using <code>grep -v "ATGC"</code>)</p>

<p>I ran the same command for other species as well and it worked fine. 
<a href="https://i.sstatic.net/jM9fz.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/jM9fz.png" alt="Example of result"></a></p>

<p>Info Update: The screenshot above was example of the result for species with TaxID 196627.</p>

<p>I wondered what is the thing that went wrong here? And what else I can check on them?</p>

CC BY-SA 4.0
2019-11-27T10:33:13.807Z
How many files match the `../com_gen/*882*.fasta` glob? Can `-subject` take more than one file as an argument? Also, aren't you mixing query and subject? What is `TBLASTNstring/882.Tstring`? That doesn't look like a fasta file, could that be the target DB name?
terdon
CC BY-SA 4.0
2019-11-27T10:42:26.450Z
`-subject` and `-query` only allow one fasta file as argument
Peter Menzel
CC BY-SA 4.0
2019-11-27T12:15:53.573Z
@PeterMenzel I haven't run blasts in ten years now. Do the modern versions really accept fasta files as `-subject`? Do they create the DB on the fly? Don't you need to first build the DB from the fasta file?
terdon
CC BY-SA 4.0
2019-11-28T01:39:21.430Z
Hi @terdon I'm sorry for not being clear enough in the description. I have just updated some info in the post which is relevant regarding your question. There is only one sequence file named `../com_gen/*882*.fasta` in my folder. (I was using * as the file name is quite long but certainly there is only 1 sequence file with the name of "882".) TBLASTNstring/882.Tstring is my query file which has more than one sequence but all in fasta format.
web
CC BY-SA 4.0
2019-11-28T01:44:49.590Z
@PeterMenzel As far as I am concerned, blast can detect all sequences in a file given if they are in fasta format? Please correct me if I am wrong.
web
CC BY-SA 4.0
2019-11-28T01:48:15.110Z
Anyway I just tried extracting one of the fasta sequences from the query file and ran it again against the genome. The same error still appear.
web
<blast><linux>
TBLASTN error: Too many positional arguments
# TBLASTN error: Too many positional arguments

(Asked by: username_0 on 2019-11-27)

I was running tblastn using standalone blast2.9.0+ through Ubuntu Linux, to get the alignment result of a list of protein sequences for same species, against its complete genome file. 
The command I was using: 
~/softwares/ncbi-blast-2.9.0+/bin/tblastn -query TBLASTNstring/882.Tstring -subject ../com_gen/*882*.fasta -outfmt "7 acc qacc pident qcovs evalue sstart send sframe" -max_hsps 1 -num_alignments 1 -out 882.blastout
(Info update: The query file has more than 1 sequence but all are in fasta format; the subject file has only 1 sequence; there is only 1 file with "882" named in the folder)
And I received error from tblastn as follow:
Error:  (CArgException::eSynopsis) Too many positional arguments (1), the offending value: ../com_gen/DP_882_DeVul_Hilde.fasta

All the sequences in both my query and subject sequence files are of two lines, the header line started with ">", followed with sequences.
I have checked both query and subject sequence files using sed -n '0~2p' | grep -v -E '[[:alpha:]]' to see if there exist special character or space within the sequence line, but the output of the results return nothing. 
PS: the complete genome file contained of nucleotide sequences with only ATGC (I have checked using grep -v "ATGC")
I ran the same command for other species as well and it worked fine. 

Info Update: The screenshot above was example of the result for species with TaxID 196627.
I wondered what is the thing that went wrong here? And what else I can check on them?

**Tags:** blast, linux
> **Comments:**
> **username_1**: How many files match the `../com_gen/*882*.fasta` glob? Can `-subject` take more than one file as an argument? Also, aren't you mixing query and subject? What is `TBLASTNstring/882.Tstring`? That doesn't look like a fasta file, could that be the target DB name?  
> **username_2**: `-subject` and `-query` only allow one fasta file as argument  
> **username_1**: @PeterMenzel I haven't run blasts in ten years now. Do the modern versions really accept fasta files as `-subject`? Do they create the DB on the fly? Don't you need to first build the DB from the fasta file?  
> **username_0**: Hi @terdon I'm sorry for not being clear enough in the description. I have just updated some info in the post which is relevant regarding your question. There is only one sequence file named `../com_gen/*882*.fasta` in my folder. (I was using * as the file name is quite long but certainly there is only 1 sequence file with the name of "882".) TBLASTNstring/882.Tstring is my query file which has more than one sequence but all in fasta format.  
> **username_0**: @PeterMenzel As far as I am concerned, blast can detect all sequences in a file given if they are in fasta format? Please correct me if I am wrong.  
> **username_0**: Anyway I just tried extracting one of the fasta sequences from the query file and ran it again against the genome. The same error still appear.  

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## 0 answers

