Paired-end RNA sequence - which read is complimentary?

Question

[yanweng]

This may be a basic question. I am doing Illumina stranded, paired-end RNA-seq, and get read1 and read2. Is read1 has the same sequence as the mRNA, or read2 has the same sequence as the mRNA?

[yanweng]

Thanks for your answers! I am using the dUTP technique, so read 2 is the same to the gene.

Answer 1

[Antonio R. Franco]

Devon was trying to tell you that if the cDNA you obtain from the RNA is stranded (using the dUTP technique) the read #2 is normally the same that the RNA. That is not the case if you made a stranded cDNA using Illumina TruSeq method (thus read # 1 is the RNA) or if your cDNA is an unstranded. In the latter case you cannot know the origen of your strands

Answer 2

[Devon Ryan]

The strand that read #2 aligns to is normally the strand from which the fragment arose. That's the case for libraries produced these days (they're all dUTP based) but wasn't always the case.

[apelin20]

Is it because we are sequencing cDNA, and cDNA is complementary to mRNA, so R1 is strand-specific to DNA which is complementary to mRNA?