A versatile method for circulating cell-free DNA methylome profiling by reduced representation bisulfite sequencing

Methylation profiling of circulating cell-free DNA (cfDNA) is of great interest as a liquid biopsy assay for the detection and monitoring of cancer and other pathologies. Here we describe circulating cell-free DNA reduced representation bisulfite sequencing (cf-RRBS), enabling the use of highly effective RRBS on fragmented plasma cfDNA. This method enriches the CpG-rich RRBS target regions by enzymatic degradation of all off-target DNA rather than by targeted capture, in contrast to previous methods. Critical steps are fully enzymatic in a single-tube, making it rapid, cost-effective, robust, and easily implemented on a liquid-handling station for high-throughput sample preparation. We benchmark cf-RRBS results to those obtained by previous more complex methods and exemplify its use for accurate non-invasive subtyping of lung cancer, a frequent onco-pathology task. cf-RRBS enables any molecular pathology lab to tap into the cfDNA methylome, only making use of off-the-shelf reagents and open-source data analysis tools. One Sentence SummaryNovel methodology enables facile and cost-effective methylation profiling of fragmented plasma DNA, allowing for routine liquid biopsy clinical diagnostics, exemplified here for differential diagnosis of lung cancer subtypes.