unpaired R1 and R2 sequences

Question

[kamiljaron]

Hello,

trimomatic reports unpaired R1 R2 after trimming pair-end reads (for those pairs where one of reads failed for some reason). Well, I kind of can not figure out, why it trimmomatic saves unpoired R1 and R2 separately. Is there any particular reason why not to concatenate those two files in one single-end reads file??

Cheers, Kamil

[Ram]

I'm taking an educated guess here, but it could be because concatenating two files is far easier than separating a file when you don't know the concatenation logic behind it?

Also, I don't think unpair-able paired end reads are the same as single end reads, although functionally they can be treated the same.

Answer 1

[Brian Bushnell]

Trimmomatic produces many output files - "forward paired", "reverse paired", R1 only, and R2 only. Personally, I think this is a huge waste of time, and paired reads should always be kept paired. BBDuk will keep paired reads paired and produce one output file for interleaved paired reads or two output files for twin input files.

If you use Trimmomatic, then what you should do with all of its output files depends on your experiment. But I suggest using BBDuk instead because the output is easier to use downstream, since it will always produce 1 (for single-ended or interleaved) or 2 (for paired in different files) files, which will be properly paired.

Answer 2

[harold.smith.tarheel]

Some tools use the read designations in the identifiers to discriminate SE from interleaved PE reads. Combining R1 and R2 of unpaired reads defeats this logic.

[kamiljaron]

I am not sure If I understand correctly. Do you mean by "designations in the identifiers" just read IDs? If yes, then the fact that they are unpaired means that there will be always only read with unique ID in the concatenated file, therefore from the perspective of the software there can not be a difference between one concatenated SE file or two R1adn R2 unpaired PE files. Right?

[harold.smith.tarheel]

Wrong. The read ID includes read1/read2 information.

Answer 3

[igor]

If the files are not interleaved before trimming, then it would make the most sense to keep them in the same format. For some libraries, R1 and R2 are not interchangeable.