Fig. 1

Flow chart representing Bisulfite Next Generation Sequencing analysis of PDCD1 and PD-L1. Formalin fixed paraffin embedded tissues were macrodissected and DNA was purified. Bisulfite treatment preceded target enrichment using primers listed in Table 2. A second low cycles PCR was then used for barcoding. Amplicons were purified by magnetic beads, quantified and pooled before loading onto MiSEQ. FASTQ files were processed for quality control (>Q30), for read lengths (>100 bp) and converted into FASTA format in a Galaxy Project environment. The methylation ratio of each CpGwas calculated using BSPAT. FFPE formalin-fixed paraffin-embedded, PCR polymerase chain reaction; BSAPT: bisulfite sequencing pattern analysis tool, mPDCD1_low low level of mPDCD1, mPDCD1_high high level of mPDCD1