Trimming adaptors and primers for RNAseq reads

Question

[Jennifer]

Hi all, I have RNA sequencing (sequenced on NextSeq 2000) reads that I know I need to cut the adaptor sequences off of and was planning to use cutadapt to do so. I have identified the adaptor sequence in my reads, but my question is do I need to cut out the index primers used during my library prep as well? I used the NebNext multiplex Oligos Index primers set 1. The primers were used in the PCR amplification of the adaptor ligated DNA. Thanks!

[ATpoint]

Adapter come before the index primer site, so if you trim adapters (in your case TruSeq adapter) that will take care of the rest as well. Fastqc will tell you whether you even have adapter contamination. Do you have any contamination?

Answer 1

[Matthias Zepper]

The FAQ of the kit is pretty clear on this ;-)

If you plan to use cutadapt, have a look at TrimGalore!, which is a convenient wrapper around it. Personally, I prefer BBDuk, see the section on Adapter trimming in the usage examples.

In case you don't know it yet: nf-core has a whole RNA-seq analysis pipeline ready for use that also takes care of the trimming.

Good look with your project Matthias

Answer 2

[dsull]

For most RNA-seq purposes, there is no need to trim adapters. Your reads will map just fine regardless of whether there's an adapter at the end.