Hello,
I am currently getting a filter length of 0 when I run the ‘filter.seqs’ command. I am using the v4 region for 16s on mothur version 1.43.0.
I am wondering if one issue has to do with the ‘pcr.seqs’ command; the ‘start’ and ‘end’ values of the reference sequence should be the same as the miseq SOP (correct)?
screen.seqs(fasta=epa19.trim.contigs.fasta, group=epa19.contigs.groups, maxambig=0, maxlength=275)
#Output File Names:
#/work/LAS/eswanner-lab/Micah/weeks9through15/epa19.trim.contigs.good.fasta
#/work/LAS/eswanner-lab/Micah/weeks9through15/epa19.trim.contigs.bad.accnos
#/work/LAS/eswanner-lab/Micah/weeks9through15/epa19.contigs.good.groups
#It took 55 secs to screen 6982343 sequences.
unique.seqs()
#Output File Names:
#/work/LAS/eswanner-lab/Micah/weeks9through15/epa19.trim.contigs.good.names
#/work/LAS/eswanner-lab/Micah/weeks9through15/epa19.trim.contigs.good.unique.fasta
count.seqs(name=epa19.trim.contigs.good.names, group=current)
#It took 156 secs to create a table for 6162489 sequences.
#Total number of sequences: 6162489
#Output File Names:
#/work/LAS/eswanner-lab/Micah/weeks9through15/epa19.trim.contigs.good.count_table
summary.seqs(count=epa19.trim.contigs.good.count_table)
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 151 151 0 3 1
2.5%-tile: 1 253 253 0 3 154063
25%-tile: 1 253 253 0 4 1540623
Median: 1 253 253 0 4 3081245
75%-tile: 1 253 253 0 5 4621867
97.5%-tile: 1 254 254 0 6 6008427
Maximum: 1 275 275 0 111 6162489
Mean: 1 252 252 0 4
# of unique seqs: 1655866
total # of seqs: 6162489
It took 41 secs to summarize 6162489 sequences.
#Output File Names:
#/work/LAS/eswanner-lab/Micah/weeks9through15/epa19.trim.contigs.good.unique.summary
pcr.seqs(fasta=silva.bacteria.fasta, start=11894, end=25319, keepdots=F, processors=8)
#[NOTE]: no sequences were bad, removing silva.bacteria.bad.accnos
#It took 4 secs to screen 14956 sequences.
#Output File Names:
#silva.bacteria.pcr.fasta
rename.file(input=silva.bacteria.pcr.fasta, new=silva.v4.fasta)
align.seqs(fasta=epa19.trim.contigs.good.unique.fasta, reference=silva.v4.fasta)
#[WARNING]: 8992 of your sequences generated alignments that eliminated too many bases, a list is provided in epa19.trim.contigs.good.unique.flip.accnos.
[NOTE]: 4731 of your sequences were reversed to produce a better alignment.
It took 585 seconds to align 1655866 sequences.
Output File Names:
epa19.trim.contigs.good.unique.align
epa19.trim.contigs.good.unique.align.report
epa19.trim.contigs.good.unique.flip.accnos
summary.seqs(fasta=epa19.trim.contigs.good.unique.align, count=epa19.trim.contigs.good.count_table)
#It took 120 secs to summarize 6162489 sequences.
#Output File Names:
#epa19.trim.contigs.good.unique.summary
filter.seqs(fasta=current, vertical=T, trump=.)
#Length of filtered alignment: 0
#Number of columns removed: 13425
#Length of the original alignment: 13425
#Number of sequences used to construct filter: 1655866
Thank you,
Micah