Abstract 529: SNAQ-SEQ™: Use of synthetic internal standards in conjunction with poisson exact test to call variants in contrived circulating tumor DNA specimens
Authors: Willey James C. , Crawford Erin , Craig Daniel J. , Xu Joshua , Haseley Nathan , Lococo Jennifer , Morrison Tom 
Published: June 2022

Abstract
Background: Targeted next generation sequencing (NGS) analysis of circulating tumor (ct) DNA promises to significantly advance targeted therapy and potentially early diagnosis of cancer. However, targeted NGS has poor accuracy for calling known variants with VAF below 0.5%. The goal of this study from the FDA-Sequencing and Quality Control Phase 2 (SEQC2) consortium was to develop and validate bioinformatic and biostatistical methods that enable incorporation of synthetic competitive internal standards (IS) in targeted NGS analysis of actionable tumor mutations.
Methods: A synthetic IS spike-in was designed for each actionable mutation target, suitable for use in NGS following targeted PCR or hybrid-capture enrichment and either with unique molecular index (UMI) or non-UMI library preparation. Contrived ctDNA reference samples developed by the SEQC2 consortium containing actionable mutations at known variant allele fraction were used. An aliquot of each sample was mixed with a mixture of IS. Following Illumina TST170 enrichment and library preparation, each library was sequenced, then native template (NT) sequences were separated from IS sequences bioinformatically. In SNAQ-SEQ™ analysis, Poisson Exact Test (PET) analysis was used to calculate the statistical difference between each sample library NT variant VAF and respective IS variant VAF. Analysis was based on NT variant count and position coverage (i.e., copies recovered in library preparation) and IS count and position coverage. PET performed an exact test of a simple null hypothesis about the ratio between two rate parameters in Poisson distribution.
Results: Stochastic sampling effect on IS error detection was minimized by ensuring an IS/NT ratio of 2.5 or greater. Under the specified conditions, in which a minimum of two NT variant observations were required for a call, the IS was able to estimate NGS background error for each sample when a minimum IS:NT ratio of 2.5:1 was used. Without use of IS information, the Illumina pipeline called 73% (41/56) of known TP variants in the 0.1% - 0.3% VAF range. In contrast, SNAQ-SEQ™ analysis (PET analysis of IS and NT information) increased TP detection sensitivity to 86% (48/56), for a 13% increase in sensitivity, with no false positives.
Conclusion: Following mixture of contrived ctDNA reference samples with IS, PET analysis enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position, in each sample. Using this SNAQ-SEQ™ analysis, true positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity. SNAQ-SEQ™ provides QC that is already standard operating procedure in clinical laboratories for analysis by high pressure liquid chromatography and mass spectrometry.
Citation Format: James C. Willey, Erin Crawford, Daniel J. Craig, Joshua Xu, Nathan Haseley, Jennifer Lococo, Tom Morrison. SNAQ-SEQ™: Use of synthetic internal standards in conjunction with poisson exact test to call variants in contrived circulating tumor DNA specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 529.