align.seqs and no of bases

Commands in mothur
1

Hello there,

I am having some issues after I run align.seqs.

mothur > summary.seqs(fasta=stability.trim.contigs.good.unique.fasta, count=stability.trim.contigs.good.count_table, processors=2)


Using 2 processors.

                Start   End     NBases  Ambigs  Polymer NumSeqs
Minimum:        1       5       5       0       1       1
2.5%-tile:      1       135     135     0       4       11367
25%-tile:       1       160     160     0       5       113670
Median:         1       229     229     0       11      227340
75%-tile:       1       229     229     0       13      341009
97.5%-tile:     1       229     229     1       20      443312
Maximum:        1       229     229     48      142     454678
Mean:   1       201.683 201.683 0.0498419       10.4269
# of unique seqs:       333146
total # of seqs:        454678

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

This is what things look like before I run align.seqs. Then…

align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=../bin/silva.bacteria/silva.bacteria.fasta)
summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table, processors=2)

Using 2 processors.

                Start   End     NBases  Ambigs  Polymer NumSeqs
Minimum:        0       0       0       0       1       1
2.5%-tile:      1044    1044    1       0       1       2782
25%-tile:       1044    1058    5       0       2       27817
Median:         6430    13125   8       0       2       55634
75%-tile:       43102   43116   11      0       2       83451
97.5%-tile:     43115   43116   160     0       10      108486
Maximum:        43116   43116   229     28      23      111267
Mean:   22362.1 23407.5 29.3862 0.0170041       2.5103
# of unique seqs:       99373
total # of seqs:        111267

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

I do not understand why after running summary.seqs the second time around, the number of bases in most of my sequences appears to be very low. Also, I appear to lose a large proportion of my sequences here. I have tried trimming the alignment using pcr.seqs, but to the same end.

Could anybody shed any light on this? Thanks in advance.

Jo

2

Have you tried running align.seqs with flip=t? If the flip parameter is set to true the reverse complement of the sequence is aligned and the better alignment is reported.

3

Hello,

In response to your advice, I re-ran align.seqs using flip=t. Unfortunately, a similar picture after running summary.seqs is evident:

mothur > summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table, processors=2)


Using 2 processors.

                Start   End     NBases  Ambigs  Polymer NumSeqs
Minimum:        0       0       0       0       1       1
2.5%-tile:      2       10      3       0       1       3231
25%-tile:       2       6699    8       0       2       32302
Median:         20727   21228   13      0       2       64604
75%-tile:       20746   21228   28      0       3       96905
97.5%-tile:     21223   21228   160     0       10      125976
Maximum:        21228   21228   229     27      23      129206
Mean:   13816.1 15055.6 32.5059 0.0220268       2.69123
# of unique seqs:       117298
total # of seqs:        129206

Any further advice would be appreciated.

Jo

4

Can you post the align.seqs command you ran to generate the align file? It looks like you used the output of pcr.seqs to generate a region-specific reference. Can you try again with silva.bacteria.fasta?

5

Hello, and sorry for my delay in replying.

I have tried align.seqs with both the output of pcr.seqs, as well as the complete alignment silva.bacteria.fasta. The output of summary.seqs remains the same.

align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=…/bin/silva.bacteria/silva.bacteria.fasta, flip=t, processors=2)

summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table, processors=2)

Using 2 processors.

                Start   End     NBases  Ambigs  Polymer NumSeqs
Minimum:        0       0       0       0       1       1
2.5%-tile:      1044    1056    4       0       1       11367
25%-tile:       1044    1067    11      0       2       113670
Median:         6428    13125   13      0       2       227340
75%-tile:       43024   43116   140     0       4       341009
97.5%-tile:     43107   43116   161     0       10      443312
Maximum:        43116   43116   229     46      74      454678
Mean:   15699.9 17915.9 58.8197 0.0351831       3.08064
# of unique seqs:       333146
total # of seqs:        454678

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

Thanks,

Jo

6

Can you post stability.trim.contigs.good.unique.fasta somewhere for us to pulldown and take a look at? Then email us at mothur.bugs@gmail and include a link to this thread. We’ll take a look and get back to you here.

Pat