Directional RNA-seq: Illumina Tru-seq versus dUTP based method

[protist]

Hi treebeard, Have you tried including actinomycin D in your second strand synthesis.

[treebeard]

follow-up: strand-specific RNA-seq

Jazz - we worked the Parkhomchuk et al. approach directly into the flow of the TruSeq mRNA-Seq kit; details are here (http://openwetware.org/wiki/Directional-RNAseq_Prep). The reported page numbers are correct for the version 1 TruSeq kit, but page numbers changed in the v2 manual (easy enough to find). We use the Illumina index adapters for multiplex sequencing (typically 6-plex), and the process works well with paired end sequencing. We suspect that there must be a small amount of carry-over of dTTP in this method, as only 95% of reads map to the expected strand (and we don't believe that the remaining 5% is due entirely to the presence of antisense RNA).

[protist]

We have been using a modified version of the Parmuchuk method (Nucleic Acids Res. 2009 Oct;37(18):e123) since 2009 with very few problems. For multiplexed samples we use 'home-brew' in-adapter indexing. I have yet to use TruSeq adapters - maybe if they brought out an oligo-only kit as they did for the original adapters we might switch...

[jazz]

Yes, I failed to mention that I needed to multiplex my samples and have paired-end reads.

[mnkyboy]

Do you want to have paired end data? Do you need to index?

We have been running the old Illumina supported directional and have had great results for whole transcriptome data. However we cannot get paired ends or multiplex this way. I tried to modify this for TruSeq and lets just say I failed miserably which they were not surprised about.

I am actually going to start working on the dUTP protocol soon and it will be interesting how it turns out.

[jazz]

I guess the title says it all here. I am looking for an RNA-seq protocol which preserves strand-specificity, and came across this paper.

Comprehensive comparative analysis of strand-specific RNA sequencing methods.
Joshua Z Levin,Moran Yassour, Xian Adiconis, Chad Nusbaum, Dawn Anne Thompson, Nir Friedman, Andreas Gnirke & Aviv Regev

http://www.nature.com/nmeth/journal/v7/n9/full/nmeth.1491.html

The authors have a done a comprehensive comparison study for various methods, and concluded that the dUTP based method (using dUTP for second strand synthesis, followed by UDG digestion) is the best among 7 others.

Since then, Illumina has come up with their Truseq RNA-seq kit.

I was wondering if anyone here has an experience to share about these 2 methods. Which one is better?

Thanks