Abstract
SUMMARYMulti-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 sub-clonal mutations by WES in the absence of orthogonal validation remains unreliable.
Highlights
A second library was generated from one of the tumor DNA samples randomly selected from each cancer and sequenced by whole-exome sequencing (WES) using the same protocol at the same facility on a different day
The validation status of 3% of the mutations could not be ascertained, because of technical failure of low AmpliSeq coverage in the validation experiments. These results suggest that WES performed at typical sequencing depth may be inadequate for detecting Intratumor genetic heterogeneity (ITGH), when the tumor cell content is less than 50%, as only 62% of the somatic mutations were detected consistently in the technical replicate pairs by WES, with the remaining mutations falsely appearing as discordant
Characteristics of Artifactual WES Somatic Mutations To identify the characteristics of the putative mutations identified by WES that were subsequently found not to be truly somatic variants, we examined the alternative coverage, the variant allele frequency (VAF), and the total depth of coverage of the candidate somatic mutations identified by the three WES pipelines from the intratumor biopsies
Summary
Intratumor genetic heterogeneity (ITGH), typically defined as the coexistence of genetically distinct but clonally related cancer cells within the same patient (Yap et al, 2012), can manifest itself spatially within the same lesion or as genetic differences between different metastatic sites and the primary tumor from the same patient (Ding et al, 2010; Gerlinger et al, 2012; Marusyk et al, 2012; Newburger et al, 2013; Yates et al, 2015). Understanding the signal-to-noise characteristics in these experiments is critical for the interpretation of ITGH
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