Must paired-end reads be in the same order in the two files for input to Tophat?

Question

[gaelgarcia]

Hi!

Will TopHat work if R1.fastq and R2.fastq have their reads in different order?

What if R1.fastq has some reads whose R2 mate did not make it past QC, and viceversa (R2.fastq has some reads whose R1 mate did not make it past QC)?

[Brian Bushnell]

Cross-posted.

Please don't post the same question on multiple forums, as it wastes people's time.

[gaelgarcia]

Noted. Thank you! I was not aware of this rule.

Answer 1

[Rob]

Paired-end reads should always be in the same order in both files when passed to an aligner. Typically, if the aligner sees a read whose mate does not appear at the same position in the other file, it will, at best, ignore that reads mate and treat the read as single-end. However, many aligners will actually complain (and possibly quit) when they encounter such a situation. Most quality-control pipelines have the ability to output reads whose mate failed QC to a separate file. The most common approach to deal with such reads is to give the aligner the QC-ed read pairs (in the exact same order in both input files) and then to provide a separate file of un-paired reads (e.g. reads whose mate failed QC). Many aligners (including Tophat, I believe) allow you to specify a set of files for paired-end reads as well as a separate file for unpaired reads in the same run.

[LayneSadler]

Same for STAR: https://groups.google.com/g/rna-star/c/NOqHwwbELCM