Which strand of the mRNA molecule does the sequencer output as a "read"?

[carmeyeii]

In Illumina Stranded RNA-Seq (using the dUTP method), do the final reads in the fastq files correspond to the initial molecule (that was transcribed), or to the reverse complement of the molecule?

C

[kmcarr]

In Illumina Stranded RNA-Seq (using the dUTP method), do the final reads in the fastq files correspond to the initial molecule (that was transcribed), or to the reverse complement of the molecule?

C

It will be different for read 1 and read 2. For the dUTP stranded protocol read 1 is the reverse complement of the mRNA, read 2 matches the sequence and direction of the mRNA. Further, read 2 should be positioned to the left (5') of read 1, when considered in transcript coordinate space.

[carmeyeii]

Thanks kmcarr,

So the strand with the same sequence and direction of the mRNA is the one retained for the first phase of paired-end sequencing, covalently linked through the P7 adapter?

C

[kmcarr]

Thanks kmcarr,

So the strand with the same sequence and direction of the mRNA is the one retained for the first phase of paired-end sequencing, covalently linked through the P7 adapter?

C

Yes, it is.

[carmeyeii]

my brain is now at rest.

thank you.