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Library construction for next-generation sequencing- Overviews and challenges

Library construction for next-generation sequencing: Overviews and challenges Steven R. Head 1, H. Kiyomi Komori 2, Sarah A. LaMere 2, Thomas Whisenant 2, Filip Van Nieuwerburgh 3, Daniel R. Salomon 2, and Phillip Ordoukhanian 11NGS and Microarray Core Facility, The Scripps Research Institute, La Jolla, CA 2Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 3Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium Abstract High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed.Keywords deep sequencing; DNA; RNA; library preparation; next-generation sequencing; RNA-seq; DNA-seq; ChIP-seq; RIP-seq

Over the past five years, next-generation sequencing (NGS) technology has become widely

available to life scientists. During this time, as sequencing technologies have improved and

evolved, so too have methods for preparing nucleic acids for sequencing and constructing

NGS libraries (1,2). For example, NGS library preparation has now been successfully demonstrated for sequencing RNA and DNA from single cells (3–11).

Address correspondence to Steven R. Head, Director, NGS and Microarray Core Facility, The Scripps Research Institute, La Jolla, CA. [email protected]

Competing Interests

The authors DRS, SRH, and PO are founding scientists and consultants to Transplant Genomics Inc.

Author contributions

SRH, PO, and DRS wrote and edited the paper. HKK contribu
ted to the Methylseq section, SALM contributed to the ChIP-seq section, TW contributed to the RIP-seq section, and FVN contributed to the sections on Nextera and mate-pair library sections.To purchase reprints of this article, contact: [email protected]://www.ypamx.icu/doc/1405f7e5a6c30c2259019efb.html

SRH, PO, and DRS wrote and edited the paper. HKK contributed to the Methylseq section, SALM contributed to the ChIP-seq section, TW contributed to the RIP-seq section, and FVN contributed to the sections on Nextera and mate-pair library sections.To purchase reprints of this article, contact: [email protected]://www.ypamx.icu/doc/1405f7e5a6c30c2259019efb.html

Library construction for next-generation sequencing- Overviews and challenges

Library construction for next-generation sequencing- Overviews and challenges

Published in final edited form as:

Biotechniques . ; 56(2): 61–passim. doi:10.2144/000114133.

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