Reversing R1.fastq and R2.fastq

Question

[L. A. Liggett]

Just as a check for the substitutions I was observing, I reversed R1.fastq and R2.fastq being the paired end reads from as sequencing experiment. Let's say at 'chr1 100' that I observe and 'A -> G' substitution. My thought here was that if I switch the paired end files so that R1.fastq becomes R2.fastq and then align and call variants, that my base substitution would now be the complimentary 'T -> C' change (using bwa mem to align and freebayes to call vars). Yet when I do this, I still get and 'A->G' change.

Am I observing no change in the substitution because of the processing by freebayes and bwa mem? Do they align to either strand and then always report the substitution on the positive strand?

[GenoMax]

Aligners will reverse complement one read from a pair when they are being aligned automatically (since the reference is only a single strand). Since most genome viewers show data with respect to "top" strand you may be observing this.

Answer 1

[mastal511]

If you just changed the names of the files without changing any of the contents, I think you should get the same alignments in both cases. For each pair of reads, one read will align to one of the strands, it doesn't matter which, and the other read will align to the opposite strand.