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Cell Rep. 2018 Nov 6;25(6):1446-1457. doi: 10.1016/j.celrep.2018.10.046.

Reliability of Whole-Exome Sequencing for Assessing Intratumor Genetic Heterogeneity.

Author information

1
Department of Medicine, Yale School of Medicine, Yale University, New Haven, CT, USA.
2
Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Institute of Pathology, University Hospital Basel, Basel, Switzerland; Department of Biomedicine, University of Basel, Basel, Switzerland.
3
Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
4
Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA; Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA.
5
Department of Medicine, Yale School of Medicine, Yale University, New Haven, CT, USA; Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA.
6
Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Institute of Pathology, University Hospital Basel, Basel, Switzerland.
7
Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA; Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA; Computer Science, Yale University, New Haven, CT, USA.
8
Department of Surgery, Yale School of Medicine, Yale University, New Haven, CT, USA; Yale Cancer Center, New Haven, CT, USA.
9
Department of Medicine, Yale School of Medicine, Yale University, New Haven, CT, USA; Yale Cancer Center, New Haven, CT, USA.
10
Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Electronic address: reisfilj@mskcc.org.
11
Department of Medicine, Yale School of Medicine, Yale University, New Haven, CT, USA; Yale Cancer Center, New Haven, CT, USA. Electronic address: christos.hatzis@yale.edu.

Abstract

Multi-region sequencing is used to detect intratumor genetic heterogeneity (ITGH) in tumors. To assess whether genuine ITGH can be distinguished from sequencing artifacts, we performed whole-exome sequencing (WES) on three anatomically distinct regions of the same tumor with technical replicates to estimate technical noise. Somatic variants were detected with three different WES pipelines and subsequently validated by high-depth amplicon sequencing. The cancer-only pipeline was unreliable, with about 69% of the identified somatic variants being false positive. Even with matched normal DNA for which 82% of the somatic variants were detected reliably, only 36%-78% were found consistently in technical replicate pairs. Overall, 34%-80% of the discordant somatic variants, which could be interpreted as ITGH, were found to constitute technical noise. Excluding mutations affecting low-mappability regions or occurring in certain mutational contexts was found to reduce artifacts, yet detection of subclonal mutations by WES in the absence of orthogonal validation remains unreliable.

KEYWORDS:

breast cancer; intratumor genetic heterogeneity; mappability; massively parallel sequencing; multi-region profiling; mutational signatures; somatic mutations; subclonal; whole-exome sequencing

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