Method for improved Illumina sequencing library preparation using NuGEN Ovation RNA-Seq System

BioTechniques

Lana Schaffer¹  G.Traver Hart¹  Daniel R. Salomon²  Phillip T. Ordoukhanian²  Steven R. Head²  John Shimashita³  H.Kiyomi Komori³ 

[1] Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA;1Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, CA, USA;;2Department of Molecular &

关键词: RNA-seq;    Next-generation sequencing;    deep sequencing;    gene expression;    cDNA library;   

DOI  :  10.2144/000113613

来源: DOAJ

【 摘 要 】

In this study, we tested the NuGEN Ovation RNA-Seq System for library preparation followed by next-generation sequencing on an Illumina GAIIx. The cDNA product of the NuGEN kit may have significant amounts of ssDNA with hairpin structures that are generated during the amplification process. These structures interfere with efficient downstream end repair, A-tailing, and adapter ligation, all standard steps in post-amplification sequencing library construction. We were able to increase the efficiency of sequencing library yields 4- to 6-fold or greater by treatment of NuGENamplified cDNA product with the single-strand endonuclease S1. These results suggest that this treatment effectively cleaves hairpin structures generated during amplification that are resistant to the standard enzyme cocktails used for the end-repair step.

【 授权许可】

Unknown