[Practical stability of whole-genome bisulfite sequencing using plasma cell-free DNA]

With the development of liquid biopsy technology, plasma cell-free DNA (cfDNA) becomes one of the research hotspots. Whole-genome bisulfite sequencing of plasma cell-free DNA has shown great potential medical applications such as cancer detection. However, the practical stability evaluation is still lacking. In this study, plasma cell-free DNA samples from two volunteers at different time were collected and prepared for sequencing in multiple laboratories. The library preparation strategies include pre-bisulfite, post-bisulfite and regular whole-genome sequencing. We established a set of quality control references for plasma cell-free DNA sequencing data and evaluated practical stability of blood collection, DNA extraction, and library preparation and sequencing depth. This work provided a technical practice guide for the application of plasma cfDNA methylation sequencing for clinical applications.