Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing

Sonia Mansukhani, Louise J Barber, Dimitrios Kleftogiannis, Sing Yu Moorcraft, Michael Davidson, Andrew Woolston, Paula Zuzanna Proszek, Beatrice Griffiths, Kerry Fenwick, Bram Herman, Nik Matthews, Ben O'Leary, Sanna Hulkki, David Gonzalez De Castro, Anisha Patel, Andrew Wotherspoon, Aleruchi Okachi, Isma Rana, Ruwaida Begum, Matthew N Davies & 10 others Thomas Powles, Katharina von Loga, Michael Hubank, Nick Turner, David Watkins, Ian Chau, David Cunningham, Stefano Lise, Naureen Starling, Marco Gerlinger

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

BACKGROUND: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies.

METHODS: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction.

RESULTS: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS, parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing.

CONCLUSIONS: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution.

LanguageEnglish
JournalClinical Chemistry
Early online date20 Sep 2018
DOIs
Publication statusEarly online date - 20 Sep 2018

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DNA Sequence Analysis
Tumors
Genes
Genome
Mutation
DNA
Colorectal Neoplasms
Neoplasms
Technology
Biopsy
Error correction
Bioinformatics
Neoplasm Genes
Hematopoiesis
Computational Biology
Point Mutation
Nucleotides
Anti-Idiotypic Antibodies
Tissue
Molecules

Cite this

Mansukhani, Sonia ; Barber, Louise J ; Kleftogiannis, Dimitrios ; Moorcraft, Sing Yu ; Davidson, Michael ; Woolston, Andrew ; Proszek, Paula Zuzanna ; Griffiths, Beatrice ; Fenwick, Kerry ; Herman, Bram ; Matthews, Nik ; O'Leary, Ben ; Hulkki, Sanna ; Gonzalez De Castro, David ; Patel, Anisha ; Wotherspoon, Andrew ; Okachi, Aleruchi ; Rana, Isma ; Begum, Ruwaida ; Davies, Matthew N ; Powles, Thomas ; von Loga, Katharina ; Hubank, Michael ; Turner, Nick ; Watkins, David ; Chau, Ian ; Cunningham, David ; Lise, Stefano ; Starling, Naureen ; Gerlinger, Marco. / Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing. In: Clinical Chemistry. 2018.
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title = "Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing",
abstract = "BACKGROUND: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies.METHODS: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction.RESULTS: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95{\%} of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100{\%} of single-nucleotide variants, with 0.15{\%} variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5{\%}. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS, parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing.CONCLUSIONS: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution.",
author = "Sonia Mansukhani and Barber, {Louise J} and Dimitrios Kleftogiannis and Moorcraft, {Sing Yu} and Michael Davidson and Andrew Woolston and Proszek, {Paula Zuzanna} and Beatrice Griffiths and Kerry Fenwick and Bram Herman and Nik Matthews and Ben O'Leary and Sanna Hulkki and {Gonzalez De Castro}, David and Anisha Patel and Andrew Wotherspoon and Aleruchi Okachi and Isma Rana and Ruwaida Begum and Davies, {Matthew N} and Thomas Powles and {von Loga}, Katharina and Michael Hubank and Nick Turner and David Watkins and Ian Chau and David Cunningham and Stefano Lise and Naureen Starling and Marco Gerlinger",
note = "{\circledC} 2018 American Association for Clinical Chemistry.",
year = "2018",
month = "9",
day = "20",
doi = "10.1373/clinchem.2018.289629",
language = "English",
journal = "Clinical Chemistry",
issn = "0009-9147",
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Mansukhani, S, Barber, LJ, Kleftogiannis, D, Moorcraft, SY, Davidson, M, Woolston, A, Proszek, PZ, Griffiths, B, Fenwick, K, Herman, B, Matthews, N, O'Leary, B, Hulkki, S, Gonzalez De Castro, D, Patel, A, Wotherspoon, A, Okachi, A, Rana, I, Begum, R, Davies, MN, Powles, T, von Loga, K, Hubank, M, Turner, N, Watkins, D, Chau, I, Cunningham, D, Lise, S, Starling, N & Gerlinger, M 2018, 'Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing', Clinical Chemistry. https://doi.org/10.1373/clinchem.2018.289629

Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing. / Mansukhani, Sonia; Barber, Louise J; Kleftogiannis, Dimitrios; Moorcraft, Sing Yu; Davidson, Michael; Woolston, Andrew; Proszek, Paula Zuzanna; Griffiths, Beatrice; Fenwick, Kerry; Herman, Bram; Matthews, Nik; O'Leary, Ben; Hulkki, Sanna; Gonzalez De Castro, David; Patel, Anisha; Wotherspoon, Andrew; Okachi, Aleruchi; Rana, Isma; Begum, Ruwaida; Davies, Matthew N; Powles, Thomas; von Loga, Katharina; Hubank, Michael; Turner, Nick; Watkins, David; Chau, Ian; Cunningham, David; Lise, Stefano; Starling, Naureen; Gerlinger, Marco.

In: Clinical Chemistry, 20.09.2018.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing

AU - Mansukhani, Sonia

AU - Barber, Louise J

AU - Kleftogiannis, Dimitrios

AU - Moorcraft, Sing Yu

AU - Davidson, Michael

AU - Woolston, Andrew

AU - Proszek, Paula Zuzanna

AU - Griffiths, Beatrice

AU - Fenwick, Kerry

AU - Herman, Bram

AU - Matthews, Nik

AU - O'Leary, Ben

AU - Hulkki, Sanna

AU - Gonzalez De Castro, David

AU - Patel, Anisha

AU - Wotherspoon, Andrew

AU - Okachi, Aleruchi

AU - Rana, Isma

AU - Begum, Ruwaida

AU - Davies, Matthew N

AU - Powles, Thomas

AU - von Loga, Katharina

AU - Hubank, Michael

AU - Turner, Nick

AU - Watkins, David

AU - Chau, Ian

AU - Cunningham, David

AU - Lise, Stefano

AU - Starling, Naureen

AU - Gerlinger, Marco

N1 - © 2018 American Association for Clinical Chemistry.

PY - 2018/9/20

Y1 - 2018/9/20

N2 - BACKGROUND: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies.METHODS: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction.RESULTS: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS, parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing.CONCLUSIONS: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution.

AB - BACKGROUND: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies.METHODS: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction.RESULTS: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS, parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing.CONCLUSIONS: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution.

U2 - 10.1373/clinchem.2018.289629

DO - 10.1373/clinchem.2018.289629

M3 - Article

JO - Clinical Chemistry

T2 - Clinical Chemistry

JF - Clinical Chemistry

SN - 0009-9147

ER -