Error during pcr.seqs step: Error in reading your fastafile, at position -1. Blank name

[sbelz]

Hello all!

I am rather new to Mothur and am looking to get some help resolving this issue. I’m using Mothur v1.48.3 and was running the pcr.seqs command:

pcr.seqs(fasta= FILENAME.trim.contigs.fasta, count= FILENAME.contigs.count_table, oligos=oligos16.txt, pdiffs=2, rdiffs=2)

and it ran for a while (several hours at least), but when I checked it this morning the last few lines of text in mothur.exe read as follows:

[..]

1727828
888427
Error in reading your fastafile, at position -1. Blank name.
496418
Error in reading your fastafile, at position -1. Blank name.
1446998
1847098

mothur >

There were no output files listed so I am inclined to believe that along with the errors, the command didn’t actually compete - though I may be wrong. If someone could offer some guidance that would be greatly appreciated.

[pschloss]

Hey -

Welcome to mothur! Can you post the commands you ran upstream of pcr.seqs? Also, what operating system are you running mothur on?

Thanks,
Pat

[sbelz]

Thank you for the reply! I am running Mothur on Windows. Here are the commands I ran leading up to this:

mothur > make.file(inputdir=mme_wd, type=fastq, prefix=mme_wd_fasta)

Output File Names:

mme_wd\mme_wd_fasta.files

mothur > make.contigs(file= mme_wd_fasta.files, processors=8)

Output File Names:

mme_wd\mme_wd_fasta.trim.contigs.fasta

mme_wd\mme_wd_fasta.scrap.contigs.fasta

mme_wd\mme_wd_fasta.contigs_report

mme_wd\mme_wd_fasta.contigs.count_table

mothur > summary.seqs(fasta= mme_wd_fasta.trim.contigs.fasta)

Output File Names:

mme_wd_fasta.trim.contigs.summary

Created a .txt file (in this case using notepad) named oligos16, copy/pasted the primer sequences listed below, and saved into the mme_wd file.

forward GTGYCAGCMGCCGCGGTAA

reverse GGACTACNVGGGTWTCTAAT

mothur > pcr.seqs(fasta= mme_wd_fasta.trim.contigs.fasta, count= mme_wd_fasta.contigs.count_table,oligos=oligos16.txt, pdiffs=2, rdiffs=2)

This is where the issue begins.

[pschloss]

Hmmm, that all seems right. Can you possibly post the content of your oligos16.txt file?

Pat

[sbelz]

So my oligos16.txt file just has the primer sequences copy pasted as written above.

This command failed before and the issue I’m posting about is what occurred when I tried to rerun the command… I am wondering at this point if I ought to just restart the pipeline? I can’t remember the issue when it first failed (it was not this same one), but I remember the Mothur.exe file being closed when it shouldn’t have been and no listing of output files.

[westcott]

The error you are getting seems to indicate an issue reading the fasta file, but the summary.seqs command is able to read it without failing. I would be happy to troubleshoot this for you. Could you send your fasta and count files to mothur.westcott@gmail.com?