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Proc Natl Acad Sci U S A. 2017 May 2;114(18):4733-4738. doi: 10.1073/pnas.1701382114. Epub 2017 Apr 17.

Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations.

Author information

1
Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21128.
2
Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21128.
3
Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21128; bertvog@gmail.com npapado1@jhmi.edu.
4
The Howard Hughes Medical Institute, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21128.
5
Department of Pathology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21128.

Abstract

The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next-generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild-type molecules.

KEYWORDS:

bisulfite sequencing; mutation; next-generation sequencing; polymerase chain reaction; strand-specificity

PMID:
28416672
PMCID:
PMC5422780
DOI:
10.1073/pnas.1701382114
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

Conflict of interest statement: N.P., K.W.K., and B.V. have no conflicts of interest with respect to the new technology described in this manuscript, as defined by the Johns Hopkins University policy on conflict of interest. N.P., K.W.K., and B.V. are founders of Personal Genome Diagnostics, Inc. and PapGene, Inc. K.W.K. and B.V. are members of the Scientific Advisory Board of Syxmex-Inostics. B.V. is also a member of the Scientific Advisory Boards of Morphotek and Exelixis GP. These companies and others have licensed technologies from Johns Hopkins University; N.P., K.W.K., and B.V. are the inventors of some of these technologies and receive equity or royalties from their licenses. The terms of these arrangements are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

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