Hi there,
mothur usually works fine for us.
We analysed our .sff data (reverse primers) with mothur v1.24.1 and it worked fine. With the same input data and mothur commands in mothur v1.25.0, we get now an error message after using the pre.cluster command:
sffinfo(sff=in.sff, flow=T)
summary.seqs(fasta=in.fasta)
trim.flows(flow=in.flow, oligos=oligos.txt, bdiffs=1, pdiffs=2, minflows=360, maxflows=720, processors=8)
shhh.flows(file=in.flow.files, processors=8)
trim.seqs(fasta=in.shhh.fasta, name=in.shhh.names, oligos=oligos.txt, flip=T, pdiffs=2, bdiffs=1, maxhomop=8, minlength=200, processors=8)
summary.seqs(fasta=in.shhh.trim.fasta, name=in.shhh.trim.names)
unique.seqs(fasta=in.shhh.trim.fasta, name=in.shhh.trim.names)
summary.seqs(fasta=in.shhh.trim.unique.fasta, name=in.shhh.trim.names)
align.seqs(fasta=in.shhh.trim.unique.fasta, reference=silva.bacteria.fasta, processors=8)
summary.seqs(fasta=in.shhh.trim.unique.align, name=in.shhh.trim.names)
screen.seqs(fasta=in.shhh.trim.unique.align, name=in.shhh.trim.names, group=in.shhh.groups, optimize=start-end, criteria=99, minlength=200, processors=8)
summary.seqs(fasta=in.shhh.trim.unique.good.align, name=in.shhh.trim.good.names)
count.groups(group=in.shhh.good.groups)
filter.seqs(fasta=in.shhh.trim.unique.good.align, vertical=T, trump=., processors=8)
unique.seqs(fasta=in.shhh.trim.unique.good.filter.fasta, name=in.shhh.trim.good.names)
pre.cluster(fasta=in.shhh.trim.unique.good.filter.unique.fasta, name=in.shhh.trim.unique.good.filter.names, group=in.shhh.good.groups, diffs=2)
missing name HB93FIC05F00A2
…
missing name HB93FIC05GNOSQ
[ERROR]: Your name file contains 28709 valid sequences, and your groupfile contains 44039, please correct.
All in all, 15330 names seem to be missing in the names file (or shoud be removed from the group file). Where did we go wrong?
We already tried to adapt the the screen.seqs command, but the error messages still popped up.
Perhaps, somebody can help us to figure out how to improve the commands in order to avoid those error messages?
Any help is highly appreciated.
Thanks a lot in advance.
Regards, stef