using the illumina 16s sequenceing primers with pcr.seqs to prepare SILVA database.

Hi all,
I am getting the error below:

mothur > align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=silva.v4.fasta)
Using 4 processors.
Reading in the silva.v4.fasta template sequences... [ERROR]: template is not aligned, aborting.
It took 0 to read  0 sequences.

I have tried to follow the MiSeq protocol, with the exception that I am using the following oligos instead of coordinates:


Those were obtained from the Illumina protocol. My workflow is as follows:

make.contigs(file=stability.files, processors=4)
screen.seqs(fasta=stability.trim.contigs.fasta, group=stability.contigs.groups, maxambig=0, maxlength=275)
count.seqs(name=stability.trim.contigs.good.names, group=stability.contigs.good.groups)
pcr.seqs(fasta=~/SILVA/silva.nr_v123.align, oligos=~/SJ_files/Lake_Magic/16s/oligos.txt,  keepdots=F, processors=4)
system(mv ~/SILVA/silva.nr_v123.pcr.align silva.v4.fasta)
align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=silva.v4.fasta)

The silva.v4.fasta file shows anout 3.2gigs of sequences, presumably the subset of the main allignment that was extracted with the primers, so that step appears to be working. Here is the summary.seqs output on silva.v4.fasta:


Using 1 processors.

  Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 16127 318 0 3 1
2.5%-tile: 2 17014 402 0 4 3342
25%-tile: 2 17014 403 0 4 33420
Median:  2 17014 422 0 5 66840
75%-tile: 2 17014 427 0 5 100260
97.5%-tile: 2 17014 429 1 7 130338
Maximum: 4399 21411 676 5 12 133679
Mean: 2.03309 17014.2 416.84 0.0475692 4.95638
# of Seqs: 133679

Output File Names: 

It took 61 secs to summarize 133679 sequences.

Any ideas where I went wrong? I also got a list of “[ERROR]: name mismatch in pcr.seqs” errors, but I was led to believe that it shouldn’t matter so long as the output file contained a reasonable number of sequences. Thanks in advance!

The keepdots parameter preserves the alignment when using the oligos parameter to trim with primers. You should set it to true.