Detection and quantification of rare mutations with massively parallel sequencing
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TLDR
An approach that can substantially increase the sensitivity of massively parallel sequencing instruments for the identification of rare variants and 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 is described.Abstract:
The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical 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. We here 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 template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with 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.read more
Citations
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Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies
Chetan Bettegowda,Chetan Bettegowda,Mark Sausen,Rebecca J. Leary,Isaac Kinde,Yuxuan Wang,Nishant Agrawal,Nishant Agrawal,Bjarne Bartlett,Bjarne Bartlett,Hao Wang,Brandon Luber,Rhoda M. Alani,Emmanuel S. Antonarakis,Nilofer S. Azad,Alberto Bardelli,Henry Brem,John L. Cameron,Clarence Lee,Leslie A. Fecher,Leslie A. Fecher,Gary L. Gallia,Peter Gibbs,Dung T. Le,Dung T. Le,Robert L. Giuntoli,Michael Goggins,Michael D. Hogarty,Matthias Holdhoff,Seung-Mo Hong,Seung-Mo Hong,Yuchen Jiao,Hartmut Juhl,Jenny J. Kim,Giulia Siravegna,Daniel A. Laheru,Calogero Lauricella,Michael Lim,Evan J. Lipson,Suely Kazue Nagahashi Marie,George J. Netto,Kelly S. Oliner,Alessandro Olivi,Louise Olsson,Gregory J. Riggins,Andrea Sartore-Bianchi,Kerstin Schmidt,le-Ming Shih,Sueli Mieko Oba-Shinjo,Salvatore Siena,Dan Theodorescu,Jeanne Tie,Timothy T. Harkins,Silvio Veronese,Tian Li Wang,Jon D. Weingart,Christopher L. Wolfgang,Laura D. Wood,Dongmei Xing,Ralph H. Hruban,Jian Wu,Peter J. Allen,C. Max Schmidt,Michael A. Choti,Victor E. Velculescu,Kenneth W. Kinzler,Bert Vogelstein,Nickolas Papadopoulos,Luis A. Diaz,Luis A. Diaz +69 more
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Analysis of Circulating Tumor DNA to Monitor Metastatic Breast Cancer
Sarah-Jane Dawson,Dana W.Y. Tsui,Muhammed Murtaza,Heather Biggs,Oscar M. Rueda,Suet-Feung Chin,Mark J Dunning,Davina Gale,Tim Forshew,Betania Mahler-Araujo,Sabrina Rajan,Sean Humphray,Jennifer Becq,David Halsall,Matthew G. Wallis,David Bentley,Carlos Caldas,Nitzan Rosenfeld +17 more
TL;DR: This proof-of-concept analysis showed that circulating tumor DNA is an informative, inherently specific, and highly sensitive biomarker of metastatic breast cancer.
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Liquid Biopsies: Genotyping Circulating Tumor DNA
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Liquid biopsies come of age: towards implementation of circulating tumour DNA
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TL;DR: The field is now in an exciting transitional period in which ctDNA analysis is beginning to be applied clinically, although there is still much to learn about the biology of cell-free DNA.
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