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

[cgini31]

Hi,
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:
Analysis/pipeline_comparison/Mothur_x_gg_13_8/gg_13_8/gg_13_8_99.pcr.fasta
Analysis/pipeline_comparison/Mothur_x_gg_13_8/gg_13_8/gg_13_8_99.bad.accnos
Analysis/pipeline_comparison/Mothur_x_gg_13_8/gg_13_8/gg_13_8_99.scrap.pcr.fasta

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

[pschloss]

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.

Pat