silva bacteria region postion

Commands in mothur
1

Hi,

I’m not sure which region of the silva reference database my primers cover, they are 27F forward and 519R, I saw a previous entry in the forum which said to run pcr.seqs on silva.bacteria.fasta using an oligos file including both the forward and revers primers and setting keepdots=T, then running summary.seqs on the output file.

I followed this procedure but my output silva.bacteria.pcr.fasta file is empty, what can I be doing wrong?

I entered this command:

mothur > pcr.seqs(fasta=silva.bacteria.fasta, oligos=pcrTest.oligos, processors=8)

Here is my oligos file:

forward AGRGTTTGATCMTGGCTCAG
reverse GTNTTACNGCGGCKGCTG
barcode CGTTTCTA N107
barcode CTAACACA N115
barcode CGTGATAA N118
barcode CGTGGGAC N119
barcode CGTCTGAA N12
barcode CGTCGCAT N3
barcode CGTGGTCA N37
barcode CGTTCACG N54
barcode CGTTTACT N56
barcode CGTGAGAC N97
barcode CGTAACCA P13
barcode CGTATTTC P18
barcode CGTAAGAA P23
barcode CGTACCCA P36
barcode CGTCAAGA P46
barcode CGTAGATA P50
barcode CGTAGGCT P61
barcode CGTCACAG P75
barcode CGTCCAGG P79
barcode CGTATTCA P9

When I run summary files:

mothur > summary.seqs(fasta=current)

I get this:

Using silva.bacteria.pcr.fasta as input file for the fasta parameter.
[ERROR]: silva.bacteria.pcr.fasta is blank, aborting.
Using silva.bacteria.pcr.fasta as input file for the fasta parameter.

Using 8 processors.
[ERROR]: silva.bacteria.pcr.fasta is blank. Please correct.
Error in reading your fastafile, at position -1. Blank name.

Thanks for your help!

Juan

2

Hi Juan,

So when we built the silva.bacteria.fasta reference we removed the 27f and 1492r primer sequences since these aren’t really sequencable (unless you do genomes). So the start position will be 1044. If you remove that from your oligos file to run pcr.seqs and then run summary.seqs you’ll get 1044 and 13127 as your start and end positions.

Pat

3

Thanks Pat, that worked great!