RNA-seq stranded protocol

Question

[rubic]

Hi,

I have RNA-seq data that was generated with Illumina TruSeq stranded protocol, according to which, as far as I understand, first mate reads are supposed to map to the same strand of the transcript and second mate reads are supposed to map to the opposite strand of the transcript. Assuming this is correct and eliminating any read-pair mappings that do not obey this condition I get very few read mappings. I noticed that I have a lot of read-pair mappings where the opposite to my condition is true. I.e., the left mate maps to the opposite strand of the transcript and the right mate maps to the same strand of the transcript. Are these read-pair mappings reliable? Should I not discard them?

[Pierre Lindenbaum]

Cross-posted: http://seqanswers.com/forums/showthread.php?t=44754

Answer 1

[Devon Ryan]

I recall that the Illumina TruSeq kit is a dUTP-based protocol, so the strand would be that of read #2.

[rubic]

So according to your answer all valid read-pair mappings should obey the rule that the first mate maps to the opposite strand and the the second mate maps to the original strand, and the any other read-pair mappings (such as the opposite pattern) should be discarded?

[Devon Ryan]

Correct