Friday, December 2, 2011

Transposome-based RNA-Seq library construction from low input amounts of RNA

A collaborative effort between Epicentre and the HudsonAlpha Institute for Biotechnology resulted in the development of two novel transposon-based methods for RNA-Seq library preparation. The technique, called Tn-RNA-Seq, can use double-stranded cDNA created from rRNA-depleted RNA to prepare an Illumina® sequencing library using only two enzymatic reactions. The researchers generated high-quality RNA-Seq libraries from as little as 10 pg of mRNA (~1 ng of total RNA) with this approach.

They also present a strand-specific RNA-Seq library construction protocol that combines transposon-based library construction with uracil DNA glycosylase and Endonuclease VIII to specifically degrade the second strand constructed during cDNA synthesis. These directional RNA-Seq libraries maintained the same quality as the nondirectional libraries, while showing a high degree of strand specificity (99.5% of reads mapped to the expected genomic strand).

A key benefit of the Tn-RNA-Seq technique is the ability to use extremely low amounts of RNA to prepare high-quality libraries. All six libraries generated using 10 ng to 10 pg of mRNA had at least 72% of aligned reads map to known transcripts, while the library made from 1 pg of mRNA had 62% of aligned reads map to known transcripts. Library complexity was found to be high for all libraries except for the library constructed with 1 pg of mRNA. In general, Tn-RNASeq libraries made with 10 pg or more of mRNA (about 50 cell equivalents) exhibited consistent quality measures. For all libraries except for the library made with 1 pg of mRNA, the rank correlations remained very high (>0.96) indicating highly consistent and reproducible library formation. The directional Tn-RNA-Seq libraries retained the same level of “strandedness” during sequencing compared to libraries made using standard adaptor-ligation methods.

The authors concluded that high-quality RNA-Seq libraries can be constructed efficiently from low input amounts of RNA using the Tn-RNA-Seq methods, and that the procedure is suitable for high-throughput or automated workflows.

ResearchBlogging.orgGertz, J. et al. (2011). Transposase mediated construction of RNA-seq libraries Genome Research DOI: 10.1101/gr.127373.111

No comments: