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Grant Details

Grant Number: 5R33CA181771-03 Interpret this number
Primary Investigator: Loeb, Lawrence
Organization: University Of Washington
Project Title: Validation and Advanced Development of Duplex Sequencing
Fiscal Year: 2016


DESCRIPTION (provided by applicant): Next generation DNA sequencing (NGS) has the power to revolutionize biology and medicine. Determining the sequence of the 3 billion nucleotides in the human genome is no longer a daunting task. However, a major impediment to the full implementation of this technology in the area of cancer detection and research is its high error rate. We have established a method that takes advantage of the complementarity inherent in double-stranded DNA. As a result of sequencing both strands of DNA we can identify true mutations as those present at the same position in both strands. Using this technology we can disregard substitutions due to PCR- amplification since they would only be present in one of the two strands. In addition, we can eliminate most sequencing errors resulting from damage to DNA templates. As a result we have establish a method that is 1000-fold more accurate than standard methods used in next generation DNA sequencing The goal of this application is to validate and further develop the method of Duplex Sequencing. Experiments will be carried out to optimize the recovery of duplex sequences and to probe the exceptional sensitivity of the methodology. To evaluate the utility of duplex DNA sequencing we will analyze hard to sequence segments of the human genome that have been resistant to sequence with accuracy using other approaches. We will also address the question of whether mutations rendering cells resistant to chemotherapeutic agents are preexistent in human cancers prior to the initiation of chemotherapy.


Immortalization of Different Breast Epithelial Cell Types Results in Distinct Mitochondrial Mutagenesis.
Authors: Kwon S. , Kim S.S. , Nebeck H.E. , Ahn E.H. .
Source: International journal of molecular sciences, 2019-06-08; 20(11), .
EPub date: 2019-06-08.
PMID: 31181796
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Detection of Low-Frequency Mutations and Identification of Heat-Induced Artifactual Mutations Using Duplex Sequencing.
Authors: Ahn E.H. , Lee S.H. .
Source: International journal of molecular sciences, 2019-01-08; 20(1), .
EPub date: 2019-01-08.
PMID: 30625989
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Single-Molecule Sequencing Reveals Patterns of Preexisting Drug Resistance That Suggest Treatment Strategies in Philadelphia-Positive Leukemias.
Authors: Schmitt M.W. , Pritchard J.R. , Leighow S.M. , Aminov B.I. , Beppu L. , Kim D.S. , Hodgson J.G. , Rivera V.M. , Loeb L.A. , Radich J.P. .
Source: Clinical cancer research : an official journal of the American Association for Cancer Research, 2018-11-01; 24(21), p. 5321-5334.
EPub date: 2018-07-24.
PMID: 30042204
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Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations.
Authors: Salk J.J. , Schmitt M.W. , Loeb L.A. .
Source: Nature reviews. Genetics, 2018 05; 19(5), p. 269-285.
EPub date: 2018-03-26.
PMID: 29576615
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Mutational spectra of aflatoxin B1 in vivo establish biomarkers of exposure for human hepatocellular carcinoma.
Authors: Chawanthayatham S. , Valentine C.C. , Fedeles B.I. , Fox E.J. , Loeb L.A. , Levine S.S. , Slocum S.L. , Wogan G.N. , Croy R.G. , Essigmann J.M. .
Source: Proceedings of the National Academy of Sciences of the United States of America, 2017-04-11; 114(15), p. E3101-E3109.
EPub date: 2017-03-28.
PMID: 28351974
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Why Cockayne syndrome patients do not get cancer despite their DNA repair deficiency.
Authors: Reid-Bayliss K.S. , Arron S.T. , Loeb L.A. , Bezrookove V. , Cleaver J.E. .
Source: Proceedings of the National Academy of Sciences of the United States of America, 2016-09-06; 113(36), p. 10151-6.
EPub date: 2016-08-19.
PMID: 27543334
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Decreased Mitochondrial Mutagenesis during Transformation of Human Breast Stem Cells into Tumorigenic Cells.
Authors: Ahn E.H. , Lee S.H. , Kim J.Y. , Chang C.C. , Loeb L.A. .
Source: Cancer research, 2016-08-01; 76(15), p. 4569-78.
EPub date: 2016-05-17.
PMID: 27197159
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The influence of subclonal resistance mutations on targeted cancer therapy.
Authors: Schmitt M.W. , Loeb L.A. , Salk J.J. .
Source: Nature reviews. Clinical oncology, 2016 06; 13(6), p. 335-47.
EPub date: 2015-10-20.
PMID: 26483300
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Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues.
Authors: Krimmel J.D. , Schmitt M.W. , Harrell M.I. , Agnew K.J. , Kennedy S.R. , Emond M.J. , Loeb L.A. , Swisher E.M. , Risques R.A. .
Source: Proceedings of the National Academy of Sciences of the United States of America, 2016-05-24; 113(21), p. 6005-10.
EPub date: 2016-05-05.
PMID: 27152024
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Human Cancers Express a Mutator Phenotype: Hypothesis, Origin, and Consequences.
Authors: Loeb L.A. .
Source: Cancer research, 2016-04-15; 76(8), p. 2057-9.
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Exploring the implications of distinct mutational signatures and mutation rates in aging and cancer.
Authors: Fox E.J. , Salk J.J. , Loeb L.A. .
Source: Genome medicine, 2016-03-17; 8(1), p. 30.
EPub date: 2016-03-17.
PMID: 26987311
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Sequencing small genomic targets with high efficiency and extreme accuracy.
Authors: Schmitt M.W. , Fox E.J. , Prindle M.J. , Reid-Bayliss K.S. , True L.D. , Radich J.P. , Loeb L.A. .
Source: Nature methods, 2015 May; 12(5), p. 423-5.
EPub date: 2015-04-06.
PMID: 25849638
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Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.
Authors: Ahn E.H. , Hirohata K. , Kohrn B.F. , Fox E.J. , Chang C.C. , Loeb L.A. .
Source: PloS one, 2015; 10(8), p. e0136216.
EPub date: 2015-08-25.
PMID: 26305705
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Detecting ultralow-frequency mutations by Duplex Sequencing.
Authors: Kennedy S.R. , Schmitt M.W. , Fox E.J. , Kohrn B.F. , Salk J.J. , Ahn E.H. , Prindle M.J. , Kuong K.J. , Shen J.C. , Risques R.A. , et al. .
Source: Nature protocols, 2014 Nov; 9(11), p. 2586-606.
EPub date: 2014-10-09.
PMID: 25299156
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Cancer: One cell at a time.
Authors: Fox E.J. , Loeb L.A. .
Source: Nature, 2014-08-14; 512(7513), p. 143-4.
EPub date: 2014-07-30.
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Accuracy of Next Generation Sequencing Platforms.
Authors: Fox E.J. , Reid-Bayliss K.S. , Emond M.J. , Loeb L.A. .
Source: Next generation, sequencing & applications, 2014; 1, .
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