Trim.seqs without .qual files

Commands in mothur
1

Hi All,

I have a bit of a problem. So I have some old data that never received the .qual files. I followed the Schloss SOP recommending to use the keepfirst command to trim my files to 200 bases. Unfortunately, when I do this, I loose all my samples when I filter using “trump=.”. When I increase the the keepfirst to 300 bases, I’m able to trim and keep the majority of the sequences in my alignment. The Schloss protocol mentions that sequence quality drops after 250 bases which is why they recommend the 200 cutoff. I’m wondering if anyone has any suggestions. Is a 300 base cutoff acceptable in this situation? Anything else I can do to get around this problem? Any advise will help. I did not try a 250 base cutoff, but I could. Thanks.

-Damon

2

Damon,

Are you running screen.seqs prior to running filter.seqs?

Pat

3

Hi Pat,

Yes, I run screen.seqs prior. I used the recommendations in your SOP screen.seqs(optimize=start, criteria=95, fasta=current, name=current). I used an “end” value just below my 2.5%-tile. It was strange since it seemed like the summary of the filtered alignment had a negative value for the sequence length.

These are archaea samples and I used the silva.archaea.fasta file as a reference. I haven’t tried the trim with my data set that is for bacteria yet. Maybe that will trim and align better? Any suggestions?

Thanks,

Damon

4

Could you send your filtered alignment file to mothur.bugs@gmail.com, so I can track down the negative values issue for you?