V3/V4 Region

Hi all,

For my sequencing, I used primers for the V3/V4 region. I’m at the pcr.seqs step of the MiSeq SOP and was wondering how I can go about searching in the SILVA reference database for where my primers start and end? Any help would be greatly appreciated!



So one option is to use a reference 16S gene (like E.coli for example) and find the region that is amplified by your primers. You can extract that region and align it against the SILVA alignment in mothur to get the start/end positions of the region you have amplified in the SILVA alignment.


Hi Richard,

Thank you for the quick response! I am still confused as to how to go about this though…

How can I find the region in the reference 16S gene? Where can I find a reference 16S gene? I have my primer sequences, but am confused as to where to go from there…Can I not align my primers sequences directly to the SILVA reference database?


you can look for your primers in the aligned file, or you can just align your seqs, run summary seq and see where to trim

I had the same problem and I found the solution in GitHub. Take a look at this link: https://github.com/mothur/mothur/issues/235

Best regards,

1 Like