Simultaneous identification of methylation and somatic variants can improve sensitivity for cancer detection and monitoring

There is increasing evidence that combining genetic and DNA methylation information in cancer research and diagnostics provides significant utility. For example, bi-allelic inactivation of tumour suppressor genes commonly occurs through a combination of somatic mutation at one allele and epigenetic silencing of the second allele and can be a key driver of cancer progression. In addition, gene fusions have an impact on DNA methylation patterns and DNA methylation changes can provide information on gene fusion status, even where the fusion event itself cannot be confidently identified in genetic sequencing data. Furthermore, combined genetic and epigenetic analysis of individual DNA fragments can increase sensitivity of somatic variant detection, which is particularly important in liquid biopsy applications. Here we analyse cell-free DNA (cfDNA) using 6-base sequencing with duet evoC, which calls genetic mutations and distinguishes between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) with high accuracy. We leverage duet evoC to confidently identify circulating-tumor DNA (ctDNA) fragments in cfDNA, a method that can be generally applied to increase the sensitivity of MRD detection. We also demonstrate that bi-allelic inactivation of tumor suppressor genes in patient samples can be identified in a single assay. Further, we identify methylation patterns that are indicative of gene fusions or chromosomal rearrangements, opening the possibility of improving the sensitivity of gene fusion detection in clinical samples.