What does IGV track color alignments by strand when using non-stranded RNA-seq?

[Villy]

I'm currently working on a RNA-seq dataset that isn't stranded, but when I load the bam files into IGV... and click on the "color alignments by strand", the majority of reads (even the ones mapped to introns) are color-coded in red or blue!

I'm not sure how it is determining which strand the read came from? How can IGV work out strand information out of nowhere, it seems?

Does anyone have a similar experience? What might be the reason?

[SylvainL]

Hi, IGV simply reports on which strand your read did map. There is absolutely no correlation with the library preparation...

[Villy]

Thanks Sylvain, but just to clarify, do you mean IGV simply reports on which cDNA strand my read did map?

And if so, how does it determine to color one cDNA (or reads mapping to that cDNA), blue; and the other one red?

[sarvidsson]

Your library preparation does not preserve strand information, so the sequences in your FASTQ files randomly correspond to either the original strand of the mRNA or the reverse complement of it. So after aligning these reads to the genome or the transcriptome, about 50 % will end up aligning to the forward strand, and about 50 % will align to the reverse strand. IGV then simply colors the reads according to the (genomic) strand the reads aligned onto...