Illumina MiSeq read orientiation

[Puhekupla]

Hi all,
I've got RNA-seq paired-end Illumina MiSeq data. I'm stuck with which read now represents the forward strand, and which one the reverse complement strand. When mapping, which read (R1/R2) should map (equals part of) to the 5' -> 3' reference sequence? And which one should map to its reverse complement?

[Header]
IEMFileVersion,4
Experiment Name,sampleA
Date,12/10/2013
Workflow,GenerateFASTQ
Application,RNA-Seq
Assay,TruSeq LT
Description,
Chemistry,Default

[mastal]

Unless the library-prep was done using a strand-specific protocol,
the reads will map to both strands.

In the Illumina system, the two reads of a pair, R1 and R2, will map to different strands.

[Puhekupla]

Yes, but which one of the pair maps to the forward reference sequence, and which one to the reverse complement? (Or should).

[relipmoc]

Yes, but which one of the pair maps to the forward reference sequence, and which one to the reverse complement? (Or should).

For each pair, one read will be mapped to one strand of the reference genome, the other will be mapped to the other strand of the reference genome (i.e. reverse complementary to the first strand). So you question can be translated into "will the first read of a pair definitely be mapped to the positive strand of the reference genome or definitely be mapped to the negative strand of the reference genome?". Of course, the answer is NO.

[bernardo_bello]

In my case:

R2 is the forward
R1 is the reverse