Allele selective enrichment for targeted profiling of rare cancer mutations via low-depth sequencing

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Molecular & Cellular Analysis Technologies
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Not Applicable
SummaryCell-free DNA (cfDNA) in peripheral blood contain tumor-derived DNA and are promising non- invasive bi-omarkers for cancer therapy selection and recurrence monitoring. However, the high cost of current commercialnext-generation sequencing (NGS) panels for profiling cancer mutations in cfDNA prevents them from beingroutinely used. Furthermore, the high sequencing depth and reads required means that cfDNA profiling is es-sentially only possible in specialized centralized reference laboratories and inaccessible to hospitals with lower-throughput sequencing instruments.Here, we propose to use allele-selective enrichment sequencing (ASES) to develop NGS panels that are capableof profiling rare cancer mutations in cfDNA with less than 0.1% variant allele frequency (VAF) using low-depthsequencing. We achieve this using allele-specific toehold probes that deplete wildtype DNA sequences and/orenrich for known mutations, increasing the VAF of rare cancer mutations by roughly 100-fold. Unlike other hy-bridization- based probes, toehold probes discriminate single nucleotide variants based on molecular competitionrather than melting temperature optimization, and thus is uniquely scalable to high multiplexing. We have ob-tained preliminary data on a NGS panel that includes 118 amplicons, and observed similar mutation sensitivityusing 98% fewer NGS reads.In this Phase I SBIR application, we will first develop an ASES NGS panel for cfDNA profiling covering 7 genesbearing 150 frequent mutations in non-small cell lung cancer patients. Subsequently, we will scale up to a 150kb hypothesis-free panel covering 50 genes.