2.4 .DNA methylation assay
Genomic DNA was extracted from peripheral blood leukocytes according to
standard procedures using a commercially available kit (E.Z.N.A.TM SQ
Blood DAN Kit) following the manufacturer’s instructions. Genomic DNA is
treated with bisulfite using EZ DNA Methylation-Gold™ Kit. Multiplex PCR
reaction was performed on the DNA after methylation. Primer information
was shown in supplementary Table 2. Products were purified using AMPure
XP beads. The Qubit 3.0 was used to determine the concentration of the
library; Agilent 2100 Bioanalyzer system was used to determine the
length of library fragments. Qualified libraries would be sequenced on
an Illumina platform. Thirty-nine CpG loci on CYP2C19 gene were
detected, and the GRCh38.p14 location information of each loci was
summarized as shown in supplementary Table 3. The level of methylation
(%methylation) is expressed as the beta value, which = methylated
cytosine/(methylated cytosine+unmethylated cytosine).