Abstract 3762: Applications of innovative whole-genome bisulfite sequencing (WGBS) for cancer biomarker discovery in plasma liquid biopsy

Abstract Circulating cell-free DNA (cfDNA) has been reported to contain valuable genetic and epigenetic information for the diagnosis and prognosis of cancer. Studies have shown that the blood from cancer patients contained more tumor-derived cfDNA compared to the healthy controls. DNA methylation, the most well-studied epigenetic marker, has been validated as one of the key drivers in the development of many diseases including cancer. cfDNA methylation thus holds great potential to become a biomarker that will enable early detection of cancer. However, cfDNA is also a challenging input for library preparation as cfDNA is mostly highly fragmented, and the amount of cfDNA from blood plasma is comparatively low. Therefore, multiple library preparation protocols were evaluated for profiling DNA methylation patterns at single nucleotide resolution from ultra-low amount of blood plasma cfDNA. These methods included two bisulfite conversion-based protocols and an enzymatic based protocol. The bisulfite conversion reaction was optimized allowing a milder treatment for less nucleic acid damage. One of the bisulfite conversion based protocols also contained a further optimized adapter ligation step, allowing a simpler procedure. Circulating cfDNA were extracted from blood plasma of a healthy control, a non-small cell lung cancer patient, and an adenocarcinoma lung cancer patient. 5-ng cfDNA was used as input and the libraries were successfully prepared using all three methods. Each library was sequenced to 300 - 600 million read pairs at a read length of 150 bp, enabling a mean coverage of about 10X per detected CpG, and over 90% of the CpG sites in human genome were detected. Unique alignment rate among all libraries were about or above 80%. The comprehensive coverage of the CpG sites across the entire human genome illustrated that the optimized bisulfite conversion reaction was compatible with fragmented cfDNA, proving the satisfactory efficacy in this simple and classic method for DNA methylation profiling of such a challenging sample type. This opens the door to apply the streamlined and simple Whole-Genome Bisulfite Sequencing (WGBS) protocol for cfDNA methylation profiling, which in turn shall facilitate further understanding in cfDNA epigenetic variations and advance the development of a cancer biomarker from the non-invasive, liquid biopsies. Citation Format: Caila Ruiz, Hanjun Kim, Zhenguo Zhang, Jeffrey Bhasin, Mingda Jin, Yi Xu, Xiaojing Yang. Applications of innovative whole-genome bisulfite sequencing (WGBS) for cancer biomarker discovery in plasma liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3762.