mothur

Length of filtered alignment: 0

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

Can you post the output that was generated to the screen when you ran

summary.seqs(fasta=epa19.trim.contigs.good.unique.align, count=epa19.trim.contigs.good.count_table)

Thanks,
Pat

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