Abstract LB-76: Absolute single-molecule sensitive NGS by SPLITSEQ: a new ctDNA analysis tool

Abstract

Abstract Circulating tumor DNA (ctDNA) analysis requires absolute single molecule sensitivity, since only a few copies of the mutation may be present per blood draw. Recent developments in next-generation sequencing (NGS) sample preparation have improved the relative sensitivity to detect <0.01% variants in background. However, when the total number of input mutant molecules falls below 10, these improvements have not translated into the absolute sensitivity required to sequence these rare templates. Here we develop and present SPLITSEQ, a novel microfluidics and NGS-based method that combines 0.01% fractional sensitivity with absolute single copy input sensitivity in an excess of background DNA. We provide multiplexed performance data on mutations in KRAS codons 12 and 13 and BRAF V600E, targets that are currently being developed as circulating predictive and prognostic biomarkers in colorectal and other cancers. Using reconstituted DNA solutions from mutant and normal cell lines ranging from 0.01% to 10% mutated allele fractions, we detected the sequence of approximately 0.2, 2, and 20 nominal mutated KRAS and BRAF inputs total in 10 000X background. Single copy numbers were supported by droplet digital PCR (ddPCR) data, an orthogonal single-molecule sensitive technique. Lastly, we confirm the specificity of the technique by screening wild-type DNA only and finding a false positive rate significantly lower than ddPCR. Moreover, in comparison with digital PCR, SPLITSEQ is easily amenable to high levels of multiplexing and detects non-recurrent mutations in tumor suppressors along the length of the sequence. Since the only requirement is a single PCR-amplifiable template, SPLITSEQ fills an unmet need in ctDNA detection, providing biomarkers from hereto unanalyzable liquid biopsies containing rare mutants. Citation Format: Ronald Lebofsky, Jordan Madic, Adrien Saliou, Francois-Clement Bidard, Olivier Lantz, Marc-Henri Stern, Jean-Yves Pierga. Absolute single-molecule sensitive NGS by SPLITSEQ: a new ctDNA analysis tool. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-76. doi:10.1158/1538-7445.AM2014-LB-76