Directional RNAseq Library Prep for NGS. How does it work? - (Jan/05/2018 )
Recently we have been looking at mRNA transcript patterns in RNA viruses using NGS. I am currently using KAPA mRNA hyperprep kit. However, some of our viral messengers are not poly-adenylated and we would like to know if a directional RNA library prep kit would work on these such samples to isolate the messenger RNA from the viral RNA.
My specific questions:
How does a directional RNA kit know which strand to get rid of?
Is there something in the analysis step which will only include RNA reads in one direction and not the other?
Will directional kits work with single-indexed libraries or must they be double-barcoded?
TruSeq libraries directional libraries are prepared by using dUTP in the second strand, regardless of polyA tail, and Script Seq (Epicentre) in a similar way. They seem to use common indexes, but you can look.
You could deplete your RNA of mRNA and rRNA and then use them on the rest, which should be your virus, in a stranded fashion.