Pcr.fasta in blank and aborts following commands after pcr.seqs

sorry if this is not the most proper section, but I need support on a step. I am using Greengenes database downloaded by your website, and using it to run mothur pipeline by following MiSeq SOP.

I am using Mothur v.1.48 in a singularity container, and working on samples that were seqeunced in MiSeq to amplify the V3-V4 region. For this reason, I try to use pcr.seqs to customize mi database, as following:

mothur > pcr.seqs(fasta=Databases/gg_13_8_otus/mothur/gg_13_8_99.refalign/gg_13_8_99.fasta, start=6388, end=13861, keepdots=F, outputdir=Analysis/pipeline_comparison/Mothur_x_gg_13_8/gg_13_8)

Command works properly, and gives me output:

It took 7 secs to screen 203452 sequences.

Output File Names:

I then proceed with rename.file, with this command:
mothur > rename.file(fasta=Analysis/pipeline_comparison/Mothur_x_gg_13_8/gg_13_8/gg_13_8_99.pcr.fasta, new=Analysis/pipeline_comparison/Mothur_x_gg_13_8/gg_13_8.v3v4.fasta, outputdir=Analysis/pipeline_comparison/Mothur_x_gg_13_8)
Setting output directory to: Analysis/pipeline_comparison/Mothur_x_gg_13_8/

And I receive this error message:

[ERROR]: Analysis/pipeline_comparison/Mothur_x_gg_13_8/gg_13_8/gg_13_8_99.pcr.fasta is blank, aborting.

Is there anything that can be done?
Thanks in advance

You are using the alignment coordinates for the SILVA reference alignment rather than for the greengenes alignment. The greengenes reference alignment is maybe ~7800 columns wide so you’re asking for an end position outside the length of the alignment. Because that doesn’t exist, I suspect that’s why your output fasta file is blank. I’d encourage you to align the greengenes database to the silva and then select the V3-V4 region with whatever coordinates you come up with for that region.


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