Length of filtered alignment=0

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**

Hi!

I’d encourage you to update your mothur version as there have been many changes since we released 1.40.5. I would also suggest you include a minlength value in your screen.seqs command. I suspect taking care of both of these issues will solve this problem.

Pat

Thank you very much for the response.

This topic was automatically closed 10 days after the last reply. New replies are no longer allowed.