Abstract 636: Accounting for processed pseudogene-related artifacts improves specificity of clinical cancer diagnostic sequencing
Authors: Artieri Carlo G. , Sikora Marcin , Artyomenko Alex , Helman Elena , Chudova Darya , Lanman Richard , Talasaz AmirAli 
Published: August 2018

Abstract
Background: Duplicated genomic regions pose a challenge to accurate variant calling in clinical sequencing applications as duplicate-specific variants may incorrectly be assigned to the target. One source of duplicated coding sequences are processed pseudogenes (PPGs) that can originate from LINE-mediated reverse transcription and genomic integration of processed mRNA, resulting in partial or complete copies of the original gene, lacking intronic sequences. False-positive variants resulting from pseudogenes found in the reference genome, such as those of PIK3CA and PTEN, have been well studied; however, the recent discovery of rare and even individual-specific cancer-related PPGs demonstrates a need for more systematic interrogation and mediation of PPG-related clinical artifacts on a sample-by-sample basis. Methods: We analyzed germline and somatic alterations in over 15,000 clinical circulating cell-free DNA samples sequenced using a 73-gene panel (Guardant Health, CA) focused primarily on clinically-relevant somatic alterations. All targeted genes were interrogated for two key hallmarks of PPGs: 1) split sequencing reads mapping to adjacent exons without intervening intronic sequence and 2) atypically increased sequencing coverage of exons relative to adjacent introns.Results: Applying a strict cutoff of ≥5 unique molecules supporting any putative intron-skipping event, we observed evidence of 94 PPGs in 91 of 15,315 analyzed samples (0.59%), affecting SMAD4 (57 samples), CDK4 (20), GNAS (6), HRAS (2), KRAS (1), TP53 (1), ESR1 (1), STK11 (1), RB1 (1), MAPK3 (1), MAPK1 (1), NF1 (1), and GATA3 (1). To determine the impact of PPGs on specificity, we applied a resampling test to assess whether somatic variant calling without PPG aware detection of SNVs results in over-representation of calls near splice junctions in samples harboring PPGs. Indeed, SMAD4 somatic SNVs were identified in 30% of samples containing SMAD4 PPGs as compared to an expected 0.7% of non-PPG samples (two-tailed p &lt; 10E-4). Similarly, 10% of samples containing CDK4 PPGs also showed somatic CDK4 SNVs, whereas only 0.9% would be expected by chance (two-tailed p = 0.03). These results strongly suggest that PPG-related artifacts may be misclassified as true somatic events in these contexts. Algorithmically restricting variant-calling only to those reads containing intronic sequence can maintain high specificity with only minimal cost to sensitivity in most exons. Conclusions: Our analysis reveals the existence of rare PPGs derived from over a dozen common clinical cancer sequencing targets and provides strong evidence that these may produce false positive SNV calls in an appreciable number of cases. Importantly, we have also shown that PPGs can be detected in a sample-specific manner based on the presence of intron-skipping split reads, enabling algorithmic detection and elimination of artifacts.
Citation Format: Carlo G. Artieri, Marcin Sikora, Alex Artyomenko, Elena Helman, Darya Chudova, Richard Lanman, AmirAli Talasaz. Accounting for processed pseudogene-related artifacts improves specificity of clinical cancer diagnostic sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 636.