Evo robot

The Infinium Methylation Assay was performed according to the manufacturer’s instructions. Briefly, 4 μl of denatured bisulfite-treated DNA was isothermally amplified over night at 37 °C, followed by an enzymatic fragmentation step. The fragmented DNA was precipitated, resuspended and loaded (using a Tecan EVO robot) on the 12-sample BeadChip, which was then incubated overnight at 48 °C, allowing the fragmented DNA to hybridize to locus-specific 50-mers. Non-specifically hybridized DNA was washed away, followed by a single-base extension reaction using DNP- and Biotin-labeled ddNTPs (with use of a Tecan EVO robot). Subsequently, hybridized DNA was removed from the labeled oligonucleotide and chips were dried under vacuum and imaged using an Illumina iScan scanner.

We also have methylation data (Illumina HumanMethylation450 DNA analysis BeadChip v1.0 Assay) from the first 125 paired tumor-normal samples (125 pairs out of the same set of 165 patients used for this study) [11 (link)]. The DNA samples were subjected to bisulfite conversion using EZ-96 DNA Methylation Kit (Zymo Research, Irvine, CA, USA). The chip presents 485,577 loci of which there are 150,254 in CpG Island, 112,067 in Shore (0–2 kb from island), 47,114 in Shelf (2–4 kb from the island), and 176,112 in deep sea (>4 kb from CpG island). We did not include the markers in the deep sea region in the final differential methylation analysis. Paired samples (CRC and corresponding normal) were processed on the same chip to avoid a batch effect. From this assay, on average 17 loci per gene were interrogated. A Tecan Evo robot was used for automated sample processing and the chips were scanned on a single iScan reader. If the intensity of the methylated loci is X and the intensity of the unmethylated loci is Y, then, the methylation score (beta value) is X/X + Y. If all are unmethylated (X = 0), then the methylation level is 0/0 + Y = 0. If all loci are methylated (Y = 0), then the beta value is X/X + 0 = 1. If 50% of probes are hybridized at the methylated loci and 50% are hybridized at the unmethylated loci, then the methylation score is 50/50 + 50 = 0.5.

We used 500 ng of 125 paired tumor and corresponding healthy tissue DNA for bisulfite conversion using an EZ-96 DNA Methylation Kit (Zymo Research, Irvine, CA, USA).
The HumanMethylation450 DNA analysis BeadChip v1.0 Assay kit was used (Illumina, San Diego, CA, USA). This chip presented 485,577 loci of which 150,254 in CpG Island, 112,067 in Shore (0–2 kb from island), 47,114 in shelf (2–4 kb from the island), and 176,112 in deep sea (>4 kb from CpG island). Paired samples (CRC and corresponding normal) were processed on the same chip to avoid the batch effect. From this assay, on average 17 loci per gene were interrogated. A Tecan Evo robot was used for automated sample processing and the chips were scanned on a single iScan reader. If the intensity of methylated loci is X and the intensity of unmethylated loci is Y, then the methylation score (beta value) is X/X + Y. If all are unmethylated (X = 0), then the methylation level is 0/0 + Y= 0. If all loci are methylated (Y = 0), then the beta value is X/X + 0= 1. If 50% probes are hybridized at methylated loci and 50% hybridized at unmethylated loci, then methylation score is 50/50 + 50 = 0.5.