Adapter trimming with trimmomatic

Question

[Mozart]

Dear all, I have a question about the use of trimmomatic. I have used the Illuminaclip step (The one which removes adapters) in trimmomatic even if the QC tool indicated no adapter contamination whatsoever. Not sure if this is an issue or not. Indeed, I wonder if using the Illuminaclip for removing Nextera or Truseq3/2 adapters, this unnecessary step would have removed fragments inside the read – should the tool not find the adapter sequence at the beginning of the read. I imagine the clipping steps are highly specific for the chosen adapters, so no harms done to the fastq files, hopefully?

Thanks

Answer 1

[GenoMax]

should the tool not find the adapter sequence at the beginning of the read.

In Illumina sequencing there should be no adapter sequences at the beginning of the read. If there is then you simply have an adapter dimer without an insert. Those need to be entirely discarded.

[Mozart]

Thanks for the speedy reply and thanks for the clarification. I wonder if adding the ILLUMINACLIP step when it is not needed it may be a problem or it is identical to the case where the ILLUMINACLIP step is not included? Hope this makes sense. Say for example I added the ILLUMINACLIP step for nextera adapters when this was not needed, would it be a problem (eg. Cut other portions or something else) or it just an unnecessary step done but the final results are exactly the same to the case where the nextera removal was not included?

[GenoMax]

Rather than the Illuminaclip, it the contents of the adapter file that you provide that influence the trimming. If you provide sequences for adapters that were not used in the library prep then that should be reflected in the data that gets trimmed i.e. there should be little or no trimming. Note: There may be some trimming just by off-chance similarity but it should be really low.