Does Cutadapt change the x,y coordinates in the fastq read name?

Question

[drkennetz]

Hi all, I have found a high presence of Illumina adapter content in a batch of samples which were sequenced on a NovaSeq. I set out to trim the adapters using cutadapt:

R1:

cutadapt -a GATCGGAAGAGCACGTCTGAACTCCAGTCAC -o outputR1.fastq.gz R1_001.fastq.gz

R2:

cutadapt -a AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT -o outputR2.fastq.gz R2_001.fastq.gz

These reads map fine with bwa mem, but I have a different type of analysis written initially for bwa backtrack (aln + sampe) and when I try to run bwa sampe it says there are mismatched read pairs (x, y coordinates are off).

If I trim the adapters with Illumina's standard bcl2fastq trimming I see some evidence that adapters were trimmed, but not to the degree I want. Cutadapt trimmed more bases, which was required in this case. I was wondering if there was a way for cutadapt to not change the x,y in the read name. Thanks

Answer 1

[GenoMax]

You should never trim PE data files independently. This causes reads to be orphaned and then the order of R1/R2 reads is messed up in resulting trimmed files. I am sure cutadapt has a PE mode which you should use with R1/R2 files. cutadapt is not changing the x,y coordinates to answer your question. Reads are getting out of sync in the two result files.

If it does not then use a different trimming program like bbduk or trimmomatic.

[drkennetz]

Thanks for the info, I have not done any kind of trimming so this has been good to know! Do you have any preference for adapter trimming software?

[GenoMax]

bbduk/trimmomatic/cutadapt/sickle etc. should all work. bbduk is easy to use but that is just a personal thing. You can find a guide for bbduk here.

[drkennetz]

Thank you! I see in the documentation that it says plainly that paired reads should always be used together (this seems like a no-brainer). Definitely a pretty bad oversight on my part.

[GenoMax]

When you care about them being in order/sync in resulting R1/R2 files. Aligners assume they are and may not always check leading to discordant mapping.