Abstract 2278: A high throughput method for the optimization of digital PCR assays for personalized circulating tumor DNA detection
Authors: Arce Maya M. , Wood-Bouwens Christina , Haslem Derrick , Lau Billy T. , Bell John , Almeda Alison , Kubit Matt , Moulton Bryce , Romero Robin , Onge Robert P. St. , Nadauld Lincoln , Ji Hanlee P. 
Published: August 2019

Abstract
Single color digital PCR (sc-dPCR) is a robust approach for the quantitation of low allelic fraction mutations in clinical oncology samples. More recently this technology has been employed to identify mutations from circulating tumor DNA (ctDNA) that has been extracted from the blood samples of cancer patients. The use of digital PCR has great potential for non-invasive longitudinal monitoring via liquid biopsies. However, this application requires low input DNA volumes and relies on a single nucleotide variant (SNV) to distinguish between normal and ctDNA, necessitating that sc-dPCR primer binding is both highly efficient and specific. These stringent requirements make assay optimization a tedious process that greatly limits the rate at which personalized detection panels can be generated. We have developed a high throughput method to optimize sc-dPCR assays utilizing Next Generation Sequencing (NGS) technology to assess amplification more quickly and with more flexibility than traditional gel based analysis.
Using our assay optimization approach, a segment of each gene containing a tumor specific SNV was incorporated into the genome of Saccharomyces cerevisiae. These renewable positive control colonies were cultured in a 96 well plate format and pooled to mimic the low allelic frequency conditions of ctDNA. The presence of each tumor specific SNV was confirmed by preparing and sequencing a library containing the unique barcode region of each colony. Using bulk PCR, up to 96 primer sets were tested at one annealing temperature in a singleplex format. Alternatively, we multiplexed up to 11 primers in each well, greatly increasing the number of assays that can be developed per plate. Using this multiplexed format, we introduced a thermal gradient across the plate to identify the optimal annealing temperature of each primer set in a single run. A parallel experiment with identical PCR conditions was run using NA18507 human DNA to act as a negative control for primer specificity.
All amplicons in each PCR condition were uniquely indexed and sequenced using an NGS platform. Using a ratio of the number of reads associated with on target and non-mutation specific amplicon sequences for each primer set, the success of each assay was determined. This method was also used to identify specific mismatches incorporated in the primer sequence that increased binding specificity. Using a sequencing based analysis method, we have observed that sc-dPCR assays can be optimized rapidly across multiple mutations, making them more accessible for personalized monitoring.
Citation Format: Maya M. Arce, Christina Wood-Bouwens, Derrick Haslem, Billy T. Lau, John Bell, Alison Almeda, Matt Kubit, Bryce Moulton, Robin Romero, Robert P. St. Onge, Lincoln Nadauld, Hanlee P. Ji. A high throughput method for the optimization of digital PCR assays for personalized circulating tumor DNA detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2278.