pcr.seqs with oligos file

Commands in mothur
1

Hi!

When I tried to customise the silva v123 to my region of interest using the primer sequences on my macbook, I only to get back 242 seqs. That is weird, right? Though the silva.nr_v123.align file looks fine to me. And some fault occurred when I tried to align my fasta to the customised silva.

a) primers sequences in pcrTest.oligos

forward GTGCCAGCMGCCGCGGTAA
reverse GGACTACHVGGGTWTCTAAT

b) summary.seqs(fasta=silva.nr_v123.align)

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1045 29691 890 0 4 1
2.5%-tile: 1046 43116 1387 0 5 4311
25%-tile: 1046 43116 1436 0 5 43105
Median: 1046 43116 1454 0 6 86210
75%-tile: 1046 43116 1470 0 6 129314
97.5%-tile: 1046 43116 1739 2 7 168108
Maximum: 1148 43116 3660 5 24 172418
Mean: 1046.01 43114.2 1474.3 0.161213 5.62284

of Seqs: 172418

c) summary.seqs(fasta=silva.nr_v123.pcr.align)

Start End NBases Ambigs Polymer NumSeqs
Minimum: 13862 23444 250 0 3 1
2.5%-tile: 13862 23444 252 0 4 7
25%-tile: 13862 23444 253 0 4 61
Median: 13862 23444 253 0 5 122
75%-tile: 13862 23444 253 0 5 182
97.5%-tile: 13862 23444 254 1 6 236
Maximum: 13862 23444 255 4 8 242
Mean: 13862 23444 252.975 0.0950413 4.76446

of Seqs: 242

d) mothur > align.seqs(fasta=CFF1.good.unique.fasta, reference=silva.nr_v123.pcr.align)

Reading in the /Users/admin/Documents/silva.nr_v123.pcr.align template sequences… DONE.
It took 0 to read 242 sequences.
Aligning sequences from /Users/admin/Documents/CFF1.good.unique.fasta …
Segmentation fault: 11
logout
Saving session…
…copying shared history…
…saving history…truncating history files…
…completed.

However, when I tried the same thing on a windows computer, I managed to get 135907 seqs back and things seem to work fine. Any idea why the difference? Problem with the mothur or silva v123 on my mac?

Thanks!

Soon

2

Sounds like it could be a memory issue. How much memory do you have on your Windows machine vs. your Mac?

3

My Mac has 4gb RAM, and my Windows has 8gb RAM. Any minimum requirement for RAM?

Thanks.

4

Mothur doesn’t have any hardcoded memory limits, but the size of the reference file will determined the amount of memory needed to run the align.seqs command. It looks like your reference file needs more than 4G and less than 8G.