Putative genes were validated using bisulfite sequencing in a validation cohort consisting of 76 samples. DNA was quantified using Picogreen (Invitrogen, California, USA) according to the manufacturer’s protocol. Briefly, 1 μg of genomic DNA was bisulfite-converted using EZ DNA Methylation according to manufacturer’s protocol (Zymo Research, California, USA). The regions of interest were amplified by PCR using a KOD-Multi & EPi (Toyobo, Osaka Japan), purified using QIAquick PCR columns (Qiagen, Venlo, Netherlands), quantified using Picogreen (Invitrogen), and verified using agarose gel electrophoresis. Libraries were prepared using an Illumina TruSeq Nano DNA sample prep kit (Illumina) according to the manufacturer’s instructions and then quantified by qPCR using a CFX96 Real-Time System (Biorad, California, USA). After normalization, the prepared library was sequenced using a Miseq system (Illumina) with 300 bp paired-end reads.
Potential sequencing adapters and low-quality bases in the raw reads were trimmed using Skewer [33 (link)] and the remaining high-quality reads were mapped to the reference genome using BS-seeker2 software [34 (link)] with a 10% mis-mapping rate. To compare the CpG methylation profiles of different sample groups, only the CpG site values were selected and the Kruskal-Wallis test was performed.
Potential sequencing adapters and low-quality bases in the raw reads were trimmed using Skewer [33 (link)] and the remaining high-quality reads were mapped to the reference genome using BS-seeker2 software [34 (link)] with a 10% mis-mapping rate. To compare the CpG methylation profiles of different sample groups, only the CpG site values were selected and the Kruskal-Wallis test was performed.
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