I am getting 0/zero as length of filtered alignment. I am working with Miseq data and I was amplifying the 16S region. Please help, what could be the problem and how can I solve it below are what I got starting from the aligning sequences.
align.seqs(fasta=Swartkops.trim.contigs.good.unique.pick.pick.fasta, reference=silva.nr_v132.align, flip=T)
Using 24 processors.
Reading in the silva.nr_v132.align template sequences... DONE.
It took 237 to read 213119 sequences.
Aligning sequences from Swartkops.trim.contigs.good.unique.pick.pick.fasta ...
It took 21805 secs to align 515503 sequences.
[WARNING]: 430 of your sequences generated alignments that eliminated too many bases, a list is provided in Swartkops.trim.contigs.good.unique.pick.pick.flip.accnos.
[NOTE]: 427 of your sequences were reversed to produce a better alignment.
Output File Names:
Swartkops.trim.contigs.good.unique.pick.pick.align
Swartkops.trim.contigs.good.unique.pick.pick.align.report
Swartkops.trim.contigs.good.unique.pick.pick.flip.accnos
For summary.seqs:
mothur > summary.seqs(fasta=Swartkops.trim.contigs.good.unique.pick.pick.align, count=Swartkops.trim.contigs.good.denovo.vsearch.pick.pick.count_table)
Using 24 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1046 1053 3 0 1 1
2.5%-tile: 11895 28464 405 0 4 26324
25%-tile: 11895 28464 409 0 4 263239
Median: 11895 28464 411 0 5 526477
75%-tile: 11895 28464 413 0 5 789715
97.5%-tile: 11895 28464 415 0 6 1026630
Maximum: 43113 43116 580 0 45 1052953
Mean: 11902 28464 410 0 4
# of unique seqs: 515503
total # of seqs: 1052953
It took 38 secs to summarize 1052953 sequences.
Output File Names:
Swartkops.trim.contigs.good.unique.pick.pick.summary
For screen.seqs:
mothur > screen.seqs(fasta=Swartkops.trim.contigs.good.unique.pick.pick.align, count=Swartkops.trim.contigs.good.denovo.vsearch.pick.pick.count_table, start=11895 end=28464)
Using 24 processors.
It took 119 secs to screen 515503 sequences, removed 9701.
/******************************************/
Running command: remove.seqs(accnos=Swartkops.trim.contigs.good.unique.pick.pick.bad.accnos, count=Swartkops.trim.contigs.good.denovo.vsearch.pick.pick.count_table)
Removed 10658 sequences from your count file.
Output File Names:
Swartkops.trim.contigs.good.denovo.vsearch.pick.pick.pick.count_table
/******************************************/
Output File Names:
Swartkops.trim.contigs.good.unique.pick.pick.good.align
Swartkops.trim.contigs.good.unique.pick.pick.bad.accnos
Swartkops.trim.contigs.good.denovo.vsearch.pick.pick.good.count_table
It took 142 secs to screen 515503 sequences.
For summary.seqs:
mothur > summary.seqs(fasta=Swartkops.trim.contigs.good.unique.pick.pick.good.align, count=Swartkops.trim.contigs.good.denovo.vsearch.pick.pick.good.count_table)
Using 24 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1046 1101 20 0 2 1
2.5%-tile: 11895 28464 405 0 4 26058
25%-tile: 11895 28464 409 0 4 260574
Median: 11895 28464 411 0 5 521148
75%-tile: 11895 28464 413 0 5 781722
97.5%-tile: 11895 28464 415 0 6 1016238
Maximum: 11895 34475 580 0 45 1042295
Mean: 11893 28463 411 0 4
# of unique seqs: 505802
total # of seqs: 1042295
It took 78 secs to summarize 1042295 sequences.
Output File Names:
Swartkops.trim.contigs.good.unique.pick.pick.good.summary
**For filter.seqs I am getting 0 as the length of filtered alignment:**
**mothur > filter.seqs(fasta=Swartkops.trim.contigs.good.unique.pick.pick.good.align, vertical=T, trump=.)**
**Using 24 processors.**
**Creating Filter... **
**It took 90 secs to create filter for 505802 sequences.**
**Running Filter... **
**It took 39 secs to filter 505802 sequences.**
**Length of filtered alignment: 0**
**Number of columns removed: 50000**
**Length of the original alignment: 50000**
**Number of sequences used to construct filter: 505802**
**Output File Names: **
**Swartkops.filter**
**Swartkops.trim.contigs.good.unique.pick.pick.good.filter.fasta**