Trim off barcodes, linker, and 16s primer sequence

Hello all,

I am new to 16s sequencing and Mothur as well. I run a 2x250bp Miseq paired ends run, amplified V4 region. Could anyone tell let me know:

  1. How to tell if my 16s raw data needs trimming of sequence adapters, barcodes, linker, and 16s primers?
  2. If trimming is necessary, how this is normally done in mothur?
  3. 16s primers are degenerate primers that contains N, V. M. Will this be a problem when it comes to trimming?
  4. How do I evaluate the quality of trimming?

Thank you very much. If similar questions have been asked in the past, any links would be appreciated.

All the best!

did you use either Caporasso or Kozich primer set up? if so, no need to trim because the primers aren’t sequenced.

Hi @kmitchell ,

Yes, I used Caporasso primer sets. It is great to know that I do not need to redo my entire analysis if trimming is not needed in my case.

I was told by sequencing center that my raw data was not trimmed off the sequencing adapters. If I do not need to trim barcode, pad, link, and primer, I should not need to trim the sequencing adapters, right? since adapters (e.g., forward adapter) are located way upstream of the primer sequence, correct?

Thank you very much. I would appreciate your confirmation.

Did you do the library prep? If not, ask your sequencing center “Did you use custom sequencing primers?”

Yes, I did the library prep, and the primer I used was exactly the same as the ones used in Caporasso paper.

Then the sequencing center must have used custom sequencing primers because the Caporasso primers do not contain any Illumina priming sites. Therefore the target primers were not sequenced.