maxambig in screen.seqs()

Commands in mothur
1

Hi,
I found a pool of my sequencing samples after make.contigs() have this kind of summary.

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

Using 1 processors.

                Start   End     NBases  Ambigs  Polymer NumSeqs
Minimum:        1 148     148     0 3 1
2.5%-tile: 1 252     252     0 4 355512
25%-tile: 1 253     253     1 4 3555113
Median:         1 253     253     1 4 7110225
75%-tile: 1 253     253     1 5 10665337
97.5%-tile:     1 254     254     3 6 13864938
Maximum:        1 302     302     125     150     14220449
Mean:   1 253.075 253.075 0.99867 4.56552
# of Seqs: 14220449

Output File Names:
full.trim.contigs.summary

May I set screen.seqs() like this

screen.seqs(fasta=full.trim.contigs.fasta, group=full.contigs.groups, maxambig=1, minlength=252, maxlength=254, maxhomop=8)

Then when I do the the last pre.cluster() with diffs=3?
Thanks in advance.

2

Your min/max length seems a bit restrictive. why don’t you follow the MiSeq SOP?

Pat

3

Actually, I really always follow the miseq_sop. For my result, most of my sequences are 252-254 bps, but they have ambig=1, only 2.5% sequences can strictly satisfy the miseq_sop requirement. Is that fine to set the ambig =1 in screen.seqs()? And set the pre.cluster(diffs=3)? I am wondering whether that makes sense during the analysis.

Thanks.

4

There is never a reason to use maxambig greater than 0. I would suggest following the command order and syntax listed in the SOP.

pat