Targeted Bisulfite Sequencing of Genomic and Circulating DNA

One microgram of genomic DNA extracted from each of the 20 tissue samples were converted following the instruction of EpiTect Fast DNA Bisulfite Kit (QIAGEN). The converted DNA was amplified using the primers (Supplementary Table 1) before sequencing. The amplification was as follows: 0.4 μM primers, 1x Uc Buffer for Library Amplification, 0.2 mM each dNTP, 1U Phanta Uc Super-Fidelity DNA polymerase. The reactions were carried out on the ProFlex™ 3 ×32-well PCR System Applied Biosystems™ with the following program: initial denaturation at 95 °C for 3 min; 35 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 60 s; then incubation at 72 °C for 7 min. The amplicons were analysed using gel electrophoresis, the band was cut and purified using QIAquick Gel Extraction Kit before the quantification with Qubit 4 Fluorometer. the PCR products were tested using Sanger sequencing. The inconsistent results were diluted 10,000 times and served as a template for nest methylated specific-PCR to generate the methylated specific-PCR product for a second round of sequencing. Sequencing data were analysed using Chromas (Technelcium Pty, v2.6.6).
Plasma cfDNA was extracted by QIAamp® Circulating Nucleic Acid Kit (Qiagen, 55114) according to the manufacturer’s protocol. DNA was subjected to the bisulfite conversion step using EZ DNA Methylation-Lightning™ Kit (Zymo Research, D5030) according to the manufacturer’s protocol.
Targeted genome methylation analysis was conducted using the OPERA Mars® Universal Library Prep Kit (Apogenomics, APG-62001), OPERA® Index Adapter (Apogenomics, APG-23005A), and OPERA® Index Primer (Apogenomics, APG-23009A) according to the manufacturer’s protocol. Briefly, the procedures were divided into three main steps: (i) multi-cycle linear amplification using a single-primer panel, (ii) ligation between amplified ssDNA product and single strand UMI adapter, which contains index sequence, (iii) indexing PCR for amplifying the ligated product with Index Primer. These target-enriched libraries were further amplified with P5 and P7 primers and purified for sequencing on the NovaSeq 6000 System (Illumina Inc).
Targeted bisulfite conversion sequence data were first trimmed by cutadapt 1.18. The reads were mapped to targets around 600-700 bp reference sequence with bismark v0.19.1, and transferred to remove duplication reads with fgbio 1.0.0 following standard instructions. The deduplicated reads were mapped to reference and the co-methylated reads were counted by manual inspection in the integrative genomics viewer (IGV v2.8.9). Reads containing more than 4 methylated CpG site was classified as co-methylated.