mothur

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.

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