Agenabio massarray iplex gold assay

For genotyping, we selected 36 susceptibility SNPs at 27 loci that had been associated with CRC risk by previous GWAS13 (link)38 (link)39 (link)40 (link)41 (link)42 (link)43 (link)44 (link)45 (link)46 (link)47 (link). For participants in the NCC 2010–2013 study, genomic DNA from blood was extracted using the MagAttract DNA Blood M48 kit and BioRobot M48 automatic extraction equipment (Qiagen, Inc., Valencia, CA, US), according to the manufacturer’s instructions. Genotyping was performed using Agenabio MassArray iPLEX^® gold assay (Agena Bioscience, Inc., San Diego, CA, US), and 32 of the 36 selected SNPs (88.9%) were successfully genotyped. Genotyping for the NCC 2000–2004 study had been conducted using the iPLEX Sequenom MassARRAY platform (Sequenom, Inc., San Diego, CA, US) for 29 susceptibility SNPs as previously described13 (link)37 (link), and 28 SNPs overlapped with the 36 SNPs selected for this analysis. Accordingly, one SNP rs719725 was excluded from the two studies. Additionally, two SNPs, rs6691170 and rs16892766, were monomorphic and therefore excluded. Thus, of the originally selected 36 SNPs, a total of 33 GWAS-identified SNPs at 25 loci were included in the analysis (Supplementary Table 1). All experimental methods were approved by the IRB of the NCC and performed in accordance with the manufacturer acguidelines and regulations.

From the National Human Genome Research Institute (NHGRI) GWAS Catalog [6 (link)], we extracted 41 CRC-associated SNPs with p-value < 5 × 10⁻⁸ reported before 2015. Among those SNPs, 14 imputed SNPs were excluded and 9 SNPs were additionally identified through reference review. The 36 susceptibility SNPs were located among 27 loci, which have been identified to be associated with CRC risk by previous GWAS. These SNPs were selected for genotyping (Additional file 2: Table S1) [15 (link)–25 (link)]. From the subjects’ blood samples, genomic DNA was extracted using a MagAttract DNA Blood M48 kit and BioRobot M48 automatic extraction equipment (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The genotyping was performed using an Agenabio MassArray iPLEX® gold assay (Agena Bioscience, Inc., San Diego, CA, US). Because of genotyping failure for 4 SNPs and a monomorphic genotype for 2 SNPs, 30 of the originally selected 36 SNPs were included in the final analysis (Additional file 2: Table S2).