Detection and quantification of rare mutations with massively parallel sequencing

Research output: Contribution to journalArticle

Abstract

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of bio-medical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. Wehere describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ("Safe-SeqS"), are (i) assignment of a unique identifier (UID) to each templatemolecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragmentswith the same UID are considered mutant ("supermutants") only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

Original languageEnglish (US)
Pages (from-to)9530-9535
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number23
DOIs
StatePublished - Jun 7 2011

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High-Throughput Nucleotide Sequencing
Mutation
Mitochondrial Genome
Biomedical Research
Polymerase Chain Reaction
DNA

Keywords

  • Biomarkers
  • Cancer
  • Diagnostics
  • Early diagnosis
  • Genetics

ASJC Scopus subject areas

  • General

Cite this

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title = "Detection and quantification of rare mutations with massively parallel sequencing",
abstract = "The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of bio-medical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. Wehere describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ({"}Safe-SeqS{"}), are (i) assignment of a unique identifier (UID) to each templatemolecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragmentswith the same UID are considered mutant ({"}supermutants{"}) only if ≥95{\%} of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.",
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AU - Kinde, Isaac

AU - Wu, Jian

AU - Papadopoulos, Nickolas

AU - Kinzler, Kenneth W

AU - Vogelstein, Bert

PY - 2011/6/7

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N2 - The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of bio-medical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. Wehere describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ("Safe-SeqS"), are (i) assignment of a unique identifier (UID) to each templatemolecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragmentswith the same UID are considered mutant ("supermutants") only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

AB - The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of bio-medical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. Wehere describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ("Safe-SeqS"), are (i) assignment of a unique identifier (UID) to each templatemolecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragmentswith the same UID are considered mutant ("supermutants") only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

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KW - Cancer

KW - Diagnostics

KW - Early diagnosis

KW - Genetics

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