Cell Reports
Volume 25, Issue 6, 6 November 2018, Pages 1446-1457
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Article
Reliability of Whole-Exome Sequencing for Assessing Intratumor Genetic Heterogeneity

https://doi.org/10.1016/j.celrep.2018.10.046Get rights and content
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Highlights

  • Genuine intratumor genetic heterogeneity is hard to distinguish from sequencing artifacts

  • Cancer-only WES pipelines are unreliable (69% somatic mutations are false positive)

  • 34%–80% of somatic variants contributing to genetic heterogeneity are technical noise

  • Excluding mutations in low-mappability regions and higher coverage are needed

Summary

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

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

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