It took me a while to figure out how to follow the instructions of pschloss.
So, I thought that I would post how to accomplish this task in a more specific manner
to further help beginners like me.
mothur “#pcr.seqs(fasta=ecoli.16srrna.fasta, oligos=pcrTest.oligos)”
A)
I obtained my ecoli 16S rRNA sequence from NCBI and saved it as a fasta file.
http://www.ncbi.nlm.nih.gov/nuccore/174375?report=fasta
B)
pcrTest.oligos is a two line file containing the primers. See http://www.mothur.org/wiki/Oligos_File for more details.
forward ACTCCTACgggAggCAgCAg
reverse GGACTACHVGGGTWTCTAAT
mothur “#align.seqs(fasta=ecoli.16srrna.pcr.fasta, reference=silva.bacteria.fasta)”
mothur “#summary.seqs(fasta=ecoli.16srrna.pcr.align)”
Results
My primers amplify the V3 and V4 regions and their 5’ positions correspond to positions 6428 and 23444 in the 50000 bp SILVA alignment. See below.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 6428 23444 429 0 6 1
2.5%-tile: 0 0 0 0 0 1
25%-tile: 0 0 0 0 0 1
Median: 0 0 0 0 0 1
75%-tile: 0 0 0 0 0 1
97.5%-tile: 0 0 0 0 0 1
Maximum: 6428 23444 429 0 6 1
Mean: 6428 23444 429 0 6
of Seqs: 1
Output File Names:
ecoli.16srrna.pcr.summary