Which Strand Of The Mrna Molecule Does The Sequencer Output As A "Read"?

Question

[gaelgarcia05]

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

[matted]

Can't you just look at a set of reads over exons where you have a good idea of the coding strand and check?

[Malachi Griffith]

I believe this post has the answer to this question, but take a look and let us know: Tophat library-type : Illumina TruSeq Stranded Total RNA Sample Prep Kit

[gaelgarcia05]

Thanks, Malachi.

My question is a bit more to do concerning the actual sequencing process - TopHat says the first strand is sequenced in the dUTP method, but if we see http://nextgen.mgh.harvard.edu/IlluminaChemistry.html , it actually makes more sense that the actual RNA molecule is what is sequenced, i.e., the first strand is used as a template for the sequencer.

If this is not so, then maybe the sequencer is actually translating the bases being incorporated into the complementary ones, so that the fastq files have the sequence of the 1st strand?

C

Answer 1

[gaelgarcia05]

It corresponds to the reverse-complementary sequence.

Now the deeper question is if the actual molecule sequenced synthesized during sequence is the sequence reported by the sequencer.