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Method for improved illumina sequencing library preparation using nugen ovation rna-seq system

Academic Article
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Overview

authors

  • Head, Steve
  • Komori-Occhicone, Heather Kiyomi
  • Hart, G. T.
  • Shimashita, J.
  • Schaffer, L.
  • Salomon, Daniel
  • Ordoukhanian, Phillip

publication date

  • March 2011

journal

  • Biotechniques  Journal

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.

subject areas

  • CD4-Positive T-Lymphocytes
  • DNA, Complementary
  • Gene Library
  • Humans
  • Polymerase Chain Reaction
  • RNA
  • Sequence Analysis, RNA
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Research

keywords

  • Next-generation sequencing
  • RNA-seq
  • cDNA library
  • deep sequencing
  • gene expression
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Identity

International Standard Serial Number (ISSN)

  • 0736-6205

Digital Object Identifier (DOI)

  • 10.2144/000113613

PubMed ID

  • 21486238
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Additional Document Info

start page

  • 177

end page

  • 180

volume

  • 50

issue

  • 3

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