Hi!
I am using mothur v.1.48.0. I used pcr.seqs with this kind of command:
pcr.seqs(fasta=testset.trim.contigs.good.fasta, count=testset.contigs.good.count_table, oligos=primers.oligos, rdiffs=1, pdiffs=1, processors=4)
When I tried pdiffs=0 and rdiffs=0, I got less sequences just as I would expect. However, when I tried pdiffs=2 and rdiffs=2, I also got LESS sequences in each sample. I would have expected that I would get more sequences when allowing larger difference with the primer sequence. I wonder why this happens?
Below are also summary.seqs in each of the three cases:
DIFFS=0
mothur > pcr.seqs(fasta=testset.trim.contigs.good.fasta, count=testset.contigs.good.count_table, oligos=primers.oligos, rdiffs=0, pdiffs=0, processors=4)
Using 4 processors.
/******************************************/
Running command: remove.seqs(accnos=testset.trim.contigs.good.bad.accnos, count=testset.contigs.good.count_table)
Removed 126786 sequences from testset.contigs.good.count_table.
Output File Names:
testset.contigs.good.pick.count_table
/******************************************/
It took 31 secs to screen 1129940 sequences.
Output File Names:
testset.trim.contigs.good.pcr.fasta
testset.trim.contigs.good.bad.accnos
testset.trim.contigs.good.scrap.pcr.fasta
testset.contigs.good.pcr.count_table
mothur > summary.seqs(fasta=current)
Using testset.trim.contigs.good.pcr.fasta as input file for the fasta parameter.
Using 4 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 99 99 0 3 1
2.5%-tile: 1 252 252 0 3 25079
25%-tile: 1 252 252 0 4 250789
Median: 1 253 253 0 4 501578
75%-tile: 1 253 253 0 5 752366
97.5%-tile: 1 254 254 0 6 978076
Maximum: 1 549 549 0 8 1003154
Mean: 1 252 252 0 4
of Seqs: 1003154
It took 4 secs to summarize 1003154 sequences.
Output File Names:
testset.trim.contigs.good.pcr.summary
mothur > count.groups(count=current)
Using testset.contigs.good.pcr.count_table as input file for the count parameter.
NW003 contains 56728.
NW004 contains 113152.
NW006 contains 207578.
NW010 contains 198100.
NW012 contains 427596.
Size of smallest group: 56728.
Total seqs: 1003154.
DIFFS=1
mothur > pcr.seqs(fasta=testset.trim.contigs.good.fasta, count=testset.contigs.good.count_table, oligos=primers.oligos, rdiffs=1, pdiffs=1, processors=4)
Using 4 processors.
/******************************************/
Running command: remove.seqs(accnos=testset.trim.contigs.good.bad.accnos, count=testset.contigs.good.count_table)
Removed 11966 sequences from testset.contigs.good.count_table.
Output File Names:
testset.contigs.good.pick.count_table
/******************************************/
It took 141 secs to screen 1129940 sequences.
Output File Names:
testset.trim.contigs.good.pcr.fasta
testset.trim.contigs.good.bad.accnos
testset.trim.contigs.good.scrap.pcr.fasta
testset.contigs.good.pcr.count_table
mothur > summary.seqs(fasta=current)
Using testset.trim.contigs.good.pcr.fasta as input file for the fasta parameter.
Using 4 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 99 99 0 3 1
2.5%-tile: 1 252 252 0 3 27950
25%-tile: 1 252 252 0 4 279494
Median: 1 253 253 0 4 558988
75%-tile: 1 253 253 0 5 838481
97.5%-tile: 1 254 254 0 6 1090025
Maximum: 1 549 549 0 8 1117974
Mean: 1 252 252 0 4
of Seqs: 1117974
It took 5 secs to summarize 1117974 sequences.
Output File Names:
testset.trim.contigs.good.pcr.summary
mothur > count.groups(count=current)
Using testset.contigs.good.pcr.count_table as input file for the count parameter.
NW003 contains 62028.
NW004 contains 140055.
NW006 contains 227243.
NW010 contains 240212.
NW012 contains 448436.
Size of smallest group: 62028.
Total seqs: 1117974.
DIFFS=2
mothur > pcr.seqs(fasta=testset.trim.contigs.good.fasta, count=testset.contigs.good.count_table, oligos=primers.oligos, rdiffs=2, pdiffs=2, processors=4)
Using 4 processors.
/******************************************/
Running command: remove.seqs(accnos=testset.trim.contigs.good.bad.accnos, count=testset.contigs.good.count_table)
Removed 68181 sequences from testset.contigs.good.count_table.
Output File Names:
testset.contigs.good.pick.count_table
/******************************************/
It took 269 secs to screen 1129940 sequences.
Output File Names:
testset.trim.contigs.good.pcr.fasta
testset.trim.contigs.good.bad.accnos
testset.trim.contigs.good.scrap.pcr.fasta
testset.contigs.good.pcr.count_table
mothur > summary.seqs(fasta=current)
Using testset.trim.contigs.good.pcr.fasta as input file for the fasta parameter.
Using 4 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 99 99 0 3 1
2.5%-tile: 1 252 252 0 3 26544
25%-tile: 1 252 252 0 4 265440
Median: 1 253 253 0 4 530880
75%-tile: 1 253 253 0 5 796320
97.5%-tile: 1 254 254 0 6 1035216
Maximum: 1 549 549 0 8 1061759
Mean: 1 252 252 0 4
of Seqs: 1061759
It took 5 secs to summarize 1061759 sequences.
Output File Names:
testset.trim.contigs.good.pcr.summary
mothur > count.groups(count=current)
Using testset.contigs.good.pcr.count_table as input file for the count parameter.
NW003 contains 60390.
NW004 contains 121622.
NW006 contains 218406.
NW010 contains 214387.
NW012 contains 446954.
Size of smallest group: 60390.
Total seqs: 1061759.