align.seqs issue/ groups removal

mothur bugs
1

please find summary results i have the number of seqs are in single digit is it normal?

also some of my groups have been completely removed again no clue why

mothur > summary.seqs(fasta=s3rfc.trim.contigs.good.unique.fasta, count=s3rfc.trim.contigs.good.count_table)

Using 1 processors.

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 265 265 0 4 1
2.5%-tile: 1 0 0 0 4 1
25%-tile: 1 268 268 0 4 2
Median: 1 268 268 0 5 3
75%-tile: 1 268 268 0 5 4
97.5%-tile: 1 268 268 0 5 5
Maximum: 1 268 268 0 5 5
Mean: 1 267.4 267.4 0 4.6

of unique seqs: 5

total # of seqs: 5

Output File Names:
s3rfc.trim.contigs.good.unique.summary


mothur > align.seqs(fasta=s3rfc.trim.contigs.good.unique.fasta, reference=silva.v5.fasta)

Using 1 processors.

Reading in the silva.v5.fasta template sequences… DONE.
It took 7 to read 14956 sequences.
Aligning sequences from s3rfc.trim.contigs.good.unique.fasta …
5
Some of you sequences generated alignments that eliminated too many bases, a list is provided in s3rfc.trim.contigs.good.unique.flip.accnos. If you set the flip parameter o true mothur will try aligning the reverse compliment as well.
It took 0 secs to align 5 sequences.


mothur > align.seqs(fasta=s3rfc.trim.contigs.good.unique.fasta, reference=silva.v5.fasta, flip=t, processors=8)

Using 8 processors.

Reading in the silva.v5.fasta template sequences… DONE.
It took 7 to read 14956 sequences.
Aligning sequences from s3rfc.trim.contigs.good.unique.fasta …
1
1
1
1
1
Some of you sequences generated alignments that eliminated too many bases, a list is provided in s3rfc.trim.contigs.good.unique.flip.accnos. If the reverse compliment provd to be better it was reported.
It took 0 secs to align 5 sequences.

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 4466 129 0 4 1
2.5%-tile: 1 4466 129 0 4 1
25%-tile: 1 4466 129 0 4 2
Median: 1 4519 129 0 4 3
75%-tile: 1239 11550 265 0 5 4
97.5%-tile: 1241 12064 268 0 5 5
Maximum: 1241 12064 268 0 5 5
Mean: 496.6 7413 184 0 4.4

of unique seqs: 5

total # of seqs: 5
mothur > screen.seqs(fasta=s3rfc.trim.contigs.good.unique.align, count=s3rfc.trim.contigs.good.count_table, summary=s3rfc.trim.contigs.good.unique.summary, start=1241, end=12064, maxhomop=8)

Using 1 processors.
Processing sequence: 5

Removing group: S3RFCBJ3 because all sequences have been removed.

Removing group: S3RFCBJ6 because all sequences have been removed.

I am sorry i am newbie (and excuse if some one has already posted the solution for such problem earlier could not find it)

thanks
sid

2

It looks like most of your sequences were removed before these commands. Possibly in a screen.seqs command? Could you post the commands you ran before these?

3

hi,
I figured out the reason for my earlier problem, no of bases were the problem…however i ran in to a new problem which i have not face before.

This time its with the chimera.uchime command, please find below
mothur > chimera.uchime(fasta=brfc.trim.contigs.good.unique.good.filter.unique.precluster.fasta, count=brfc.trim.contigs.good.unique.good.filter.unique.precluster.count_table, dereplicate=t)

Using 1 processors.
uchime by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.

Checking sequences from brfc.trim.contigs.good.unique.good.filter.unique.precluster.fasta …
uchime v4.2.40
by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.

00:00 18Mb 0.1% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
WARNING: Ignoring gaps in FASTA file ‘brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp’
00:00 18Mb 100.0% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
00:00 18Mb 75 sequences
00:00 1.9Mb 100.0% 0/74 chimeras found (0.0%)

It took 0 secs to check 75 sequences from group BRFCBA3.
uchime v4.2.40
by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.

00:00 18Mb 0.1% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
WARNING: Ignoring gaps in FASTA file ‘brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp’
00:00 18Mb 100.0% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
00:00 18Mb 47 sequences
00:00 1.9Mb 100.0% 0/46 chimeras found (0.0%)

It took 0 secs to check 47 sequences from group BRFCBA4.
uchime v4.2.40
by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.

00:00 18Mb 0.1% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
WARNING: Ignoring gaps in FASTA file ‘brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp’
00:00 18Mb 100.0% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
00:00 18Mb 88 sequences
00:00 1.8Mb 100.0% 0/87 chimeras found (0.0%)

It took 0 secs to check 88 sequences from group BRFCU1.
uchime v4.2.40
by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.

00:00 18Mb 0.1% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
WARNING: Ignoring gaps in FASTA file ‘brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp’
00:00 18Mb 100.0% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
00:00 18Mb 115 sequences
00:00 1.8Mb 100.0% 0/114 chimeras found (0.0%)

It took 0 secs to check 115 sequences from group BRFCU2.
uchime v4.2.40
by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.

00:00 18Mb 0.1% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
WARNING: Ignoring gaps in FASTA file ‘brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp’
00:00 18Mb 100.0% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
00:00 18Mb 110 sequences
00:00 1.8Mb 100.0% 0/109 chimeras found (0.0%)

It took 0 secs to check 110 sequences from group BRFCU6.
uchime v4.2.40
by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.

00:00 18Mb 0.1% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
WARNING: Ignoring gaps in FASTA file ‘brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp’
00:00 18Mb 100.0% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
00:00 18Mb 114 sequences
00:00 1.8Mb 100.0% 0/113 chimeras found (0.0%)

It took 0 secs to check 114 sequences from group BRFCU7.
uchime v4.2.40
by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.

00:00 18Mb 0.1% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
WARNING: Ignoring gaps in FASTA file ‘brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp’
00:00 18Mb 100.0% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
00:00 18Mb 120 sequences
00:00 1.8Mb 100.0% 0/119 chimeras found (0.0%)

It took 0 secs to check 120 sequences from group BRFCX2.
uchime v4.2.40
by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.

00:00 18Mb 0.1% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
WARNING: Ignoring gaps in FASTA file ‘brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp’
00:00 18Mb 100.0% Reading brfc.trim.contigs.good.unique.good.filter.unique.precluster.temp
00:00 18Mb 123 sequences
00:00 1.8Mb 100.0% 0/122 chimeras found (0.0%)

It took 0 secs to check 123 sequences from group BRFCX3.
[ERROR]: brfc.trim.contigs.good.unique.good.filter.unique.precluster.uchime.accnos is blank. Please correct.

Output File Names:
brfc.trim.contigs.good.unique.good.filter.unique.precluster.uchime.pick.count_table
brfc.trim.contigs.good.unique.good.filter.unique.precluster.uchime.chimeras
brfc.trim.contigs.good.unique.good.filter.unique.precluster.uchime.accnos

How can it be blank? when i set dereplicate=F the following is the result
It took 0 secs to check 123 sequences from group BRFCX3.

It took 1 secs to check 792 sequences. 0 chimeras were found.
The number of sequences checked may be larger than the number of unique sequences because some sequences are found in several samples.

Output File Names:
brfc.trim.contigs.good.unique.good.filter.unique.precluster.uchime.chimeras
brfc.trim.contigs.good.unique.good.filter.unique.precluster.uchime.accnos

Best,
Sid

4

Uchime is reporting 0 chimeras across all your groups.

5

yes true but how do i proceed with the pipeline, the next command is remove.seqs which I am unable to run as accnos is blank !
should i directly run classify.seqs?

Thanks.

Best,
Sid

6

You do not need to run the remove.seqs command because there are no chimeras to remove. You can proceed with the pipeline.