title: Method for improved Illumina sequencing library preparation using NuGEN Ovation RNA-Seq System.
abstract: 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 NuGEN-amplified 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.
authors.affiliation: Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, CA 92040, USA. shead@scripps.edu
authors.forename: Steven R
authors.initials: SR
authors.lastname: Head
authors.forename: H Kiyomi
authors.initials: HK
authors.lastname: Komori
authors.forename: G Traver
authors.initials: GT
authors.lastname: Hart
authors.forename: John
authors.initials: J
authors.lastname: Shimashita
authors.forename: Lana
authors.initials: L
authors.lastname: Schaffer
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Salomon
authors.forename: Phillip T
authors.initials: PT
authors.lastname: Ordoukhanian
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000191:economics / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D012313:RNA