10.2. How to trim 16S rRNA primers of paired Illumina reads?
This FAQ describes how to trim the 16S rRNA primers from 2 x 301 bp Illumina paired reads prepared using a stranded protocol.
If the reads have been prepared with a stranded protocol sequencing the top strand of the 16S rRNA amplicon, then the forward primer will be present in the 5' end of Read 1 and the reverse primer in the 5' end of Read 2. An example of this using the Bakt_341F 5′-CCTACGGGNGGCWGCAG-3′ and Bakt_805R 5′-GACTACHVGGGTATCTAATCC-3 primers generating an approx. 450 bp long amplicons which covers the variable regions 3–4 is found below:
To trim such primers, you can create a Trim adapter list as shown on the image below:
In the Trim Adapter List, the read to trim have been specified. Specifying the read to trim allows the option to discard reads in which the expected primer is not found. This is recommended as such reads might be of low quality or contamination of the sample.
Since, the full primer sequence is found in the 5'end of the read it can be recognized for trimming both as an internal and end match. You may wish to optimize the match thresholds for your adapters. How to create a Trim Adapter List and set thresholds for when a match is found is described in the manual pages as follows:
If you are unsure which protocol have been used for generation of your reads, then you may wish to check the location and orientation of the primers in the reads. How to do this is described in the related FAQ.