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

Steven R. Head; H. Kiyomi Komori; G. Traver Hart; John Shimashita; Lana Schaffer; Daniel R. Salomon; Phillip T. Ordoukhanian

doi:10.2144/000113613

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

Steven R. Head, H. Kiyomi Komori, G. Traver Hart, John Shimashita, Lana Schaffer, Daniel R. Salomon, Phillip T. Ordoukhanian

Research output: Contribution to journal › Article › peer-review

25 Scopus citations

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 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.

Original language	English (US)
Pages (from-to)	177-181
Number of pages	5
Journal	BioTechniques
Volume	50
Issue number	3
DOIs	https://doi.org/10.2144/000113613
State	Published - Mar 2011
Externally published	Yes

Keywords

Deep sequencing
Gene expression
Next-generation sequencing
RNA-seq
cDNA library

ASJC Scopus subject areas

Biotechnology
General Biochemistry, Genetics and Molecular Biology

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10.2144/000113613

Cite this

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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 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.",

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AU - Traver Hart, G.

AU - Shimashita, John

AU - Schaffer, Lana

AU - Salomon, Daniel R.

AU - Ordoukhanian, Phillip T.

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KW - Next-generation sequencing

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

Abstract

Keywords

ASJC Scopus subject areas

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