I am using the Miseq SOP to analyze 18S sequences and using Silva 132 for alignment and classification. I’m looking at the v9 region using Earth Microbiome primers 1391f and EukB, expected amplicon length around 260+/-50. After creating a reference alignment to analyze my mock community, it looks like the sequence itself, including the primers, is about 174 bp (close to the published length of 168bp for S. cerevisiae, Stoek et al 2010, cited on Earth Microbiome website in reference to these primers).
I am trying to use an S. cerevisiae sequence from that reference alignment and follow the mothur “customize you reference alignment” protocol for 16S data, as listed below. However, the summary table I’m getting is all zeroes. What am I missing here?
Any help greatly appreciated!
Sequence in Scerevisiae_v9.fasta:
Reverse Complement of Reverse Primer GTAGGTGAACCTGCAGAAGGATCA