Nanopore Sequencing of CpG and GpC Methylation Training and Testing Samples

Genomic DNA from E. coli K12 MG1655 (ATCC 700926DQ) and NA12878 (genomic DNA from GM12878 lymphoblast cell line, Coriell Institute) were first sheared to an average fragment size of 8 kb using g-tubes (Covaris Cat. 520079). The fragmented DNA wasPCR amplified to generate unmethylated DNA using the first steps of low i nput ligation kit SQK-LWP001 (ONT). Samples were end-repaired, deoxyadenosine(dA)-tailed, and ligated to amplification adaptors, followed by 11 cycles of PCR amplification. The resulting unmethylated, sheared DNA was methylated with M. SssI (NEB Cat. M0226) for CpG methylation or M. CviPI (NEB Cat. M0227) for GpC methylation, or both enzymes for CpG+GpC methylation. Two cycles of 4-hour methylation were performed for each sample, and for each cycle of treatment the enzyme and methyl donor (S-adenosylmethionine) were replenished at the 2 hour mark. The resulting set of DNA samples were subjected to nanopore sequencing using the ligation sequencing kit LSK-SQK-108 (ONT).