Abstract 6609: Detection of Rare Mutations, Copy Number Variation, and DNA Methylation in the Same Template DNA Molecules

The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To increase the sensitivity of such assays, it would be useful to be able evaluate the same template molecules for all these changes. Here we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The original and copied strands are queried for epigenetic and genetic alterations, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung or colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics in the future. Citation Format: Yuxuan Wang, Christopher Douville, Joshua Cohen, Chetan Bettegowda, Nick Papadopoulos, Ken Kinzler, Bert Vogelstein. Detection of rare mutations, copy number variation, and DNA methylation in the same template DNA molecules. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6609.