IGV visualization pattern for RNA-Seq mapping

Question

[blz]

Hello everyone,

When I look at RNA-Seq mapping in IGV, why reads are aligned to the opposite strand?

In the example below, the gene is annotated in the reverse strand (which I understand as a synonym of negative/minus strand), but the reads are aligned to the positive strand (foward/plus strand). It's because is cDNA? (I used Group alignments by first-in-pair strand and Color alignments by first-of-pair strand options in IGV)

Sorry for the silly question, but I could not figured out it by myself...

Many thanks.

[liorglic]

I think this has to do with the RNA-seq library prep protocol. There are "stranded" protocols that will always produce reads from the +/- strand. See this for example.

[blz]

Yes, it's a strand specific protocol (dUTP).

But this is not an exception, this is the rule: for all genes I've seen, the reads are aligned to the opposite strand.

Maybe should I use strand info when mapping (i.e. in bowtie2)? Or I'm using wrong options in IGV (Group alignments by first-in-pair strand and Color alignments by first-of-pair strand)?

[liorglic]

Maybe I'm confused here, but doesn't that simply mean that your protocol is minus-strand specific? You can check that by running your aligned bam file through Qualimap. The report will tell you the proportion of +/- alignments.

[blz]

I ran infer_experiment.py (from RSeQC package) and the results pointed to RF orientation of pairs, which I think you mean "minus-strand specific".

This is PairEnd Data
Fraction of reads failed to determine: 0.0000
Fraction of reads explained by "1++,1--,2+-,2-+": 0.0005
Fraction of reads explained by "1+-,1-+,2++,2--": 0.9995

[jv]

if your data is paired end, then (depending on the protocol) one read of the pair will be sense orientation and the other read will be antisense orientation. See https://www.biostars.org/p/285757/ for more details

Answer 1

[blz]

But I still can not understand why the first read is in the reverse complementary of transcript strand...

Considering the following image (from https://github.com/igordot/genomics/blob/master/notes/rna-seq-strand.md):

the first cDNA strand synthesized is used to sequencing, so the first read should not be equivalent to mRNA strand (and, therefore, the first read should rely on the transcript strand)?

Many thanks!

Answer 2

[blz]

Maybe now I can understand...

In the schematic representation below,

suppose the red read is the first read of the pair.

The red read is in the reverse complementary of transcript strand.

Then, when I Group alignments by first-in-pair strand in IGV, I'm grouping reads by the strand of the red read, which is the reverse complementary of transcript strand. And the transcript strand is the opposite strand.