I’ve gone through several Mothur runs successfully incorporating the following chimera removal step as found in the Miseq SOP
chimera.vsearch(fasta=current, count=current, dereplicate=t)
remove.seqs(fasta=current, accnos=current)
I want to use a reference based chimera removal instead.
chimera.vsearch(fasta=current, reference=SymRef3.fasta)
remove.seqs(fasta=current, accnos=current)
I noticed that the output said that 0 sequences were removed. I pulled out the relative files and ran this particular part:
mothur > remove.seqs(fasta=stabilityps1.trim.contigs.good.unique.good.filter.unique.precluster.fasta, accnos=stabilityps1.trim.contigs.good.unique.good.filter.unique.precluster.ref.vsearch.accnos) Removed 0 sequences from your fasta file.
Output File Names:
stabilityps1.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta
mothur > summary.seqs(count=current) [WARNING]: no file was saved for count parameter. Using stabilityps1.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta as input file for the fasta parameter.
Using 1 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 846 401 0 3 1
2.5%-tile: 1 846 401 0 4 3262
25%-tile: 1 846 425 0 4 32620
Median: 1 846 425 0 4 65240
75%-tile: 1 846 425 0 5 97860
97.5%-tile: 1 846 425 0 6 127218
Maximum: 1 846 425 0 8 130479
Mean: 1 846 421.131 0 4.47999
of Seqs: 130479
Output File Names:
stabilityps1.trim.contigs.good.unique.good.filter.unique.precluster.pick.summary
I searched for" M02740_42_000000000-AFFVB_1_1101_6316_305" the first accnos found in stabilityps1.trim.contigs.good.unique.good.filter.unique.precluster.ref.vsearch.accnos and found it in stabilityps1.trim.contigs.good.unique.good.filter.unique.precluster.fasta.
I don’t know why remove.seqs(fasta=current, accnos=current) doesn’t work if I used a reference for the chimera search but the same commands for remove.seqs do work if the chimera search was done de novo (chimera.vsearch(fasta=current, count=current, dereplicate=t)).