trim.flows in version 1.32 producing empty flow.files

mothur bugs
1

Hi, I have separate sff files for each of my samples and I am trying to work on them separately until I get to the unique.seqs step.
When I run
mothur > trim.flows(flow=sample.flow, oligos=sample.oligos, pdiffs=2, bdiffs=1)

Output File Names:
sample.trim.flow
sample.scrap.flow
sample.flow.files

I run the shhh.flows command and obtain the following error message:
[ERROR]: sample.flow.files is blank, aborting.
values for either flow or file must be provided for the shhh.flows command.
Unable to open ~/LookUp_Titanium.pat. Trying mothur’s executable location ~/LookUp_Titanium.pat
Unable to open ~/LookUp_Titanium.pat.

When I checked the sample.scrap.flow file I saw in the first row 450 and beneath that sequenceID|lbf or sequenceID|bf

Any idea what the problem might be?

Thanks,

Claudia

2

Are there any sequenceIDS in sample.trim.flow?

3

Nope, only the number of flows that I selected. In my case, as I have tried with 450 and 360, these numbers are the only thing I see in two different trim.flow files.

4

If the *.trim.flow file is empty then all your sequences are being scrapped. From the scrap codes you posted, it looks like they are failing due to barcodes and primers. Perhaps its a formatting issue on the oligos file? Can you post your oligos file?

5

Yep! It was an issue with the barcodes. “Fortunately” the main problem was that the barcodes provided by the core facility where I sent my samples were nor the correct ones. That is why in the scrap.flow file all the sequences showed|b.

However, I still have a considerable amount of sequence in the scrap.flow file that were filtered by length (I see lots of |l). If I am using the primers 28F/519R for bacteria and 349F/806R for archaea in a GS FLX Titanium platform. In that case, is it save to use 450 as the number of flowgrams in trim.flow?

Thanks!