Genotyping console 4

DNA labelling, hybridization, and array scanning were performed for SNP 6.0 arrays (Affymetrix, Santa Clara, CA) according to the manufacturer's protocol and processed with the Affymetrix Genotyping Console 4.0 software to generate Birdseed .chp and .txt files using the Birdseed v2 algorithm with the default parameters., Model-based expression analysis was performed and imported into R for reference normalization to a diploid reference chromosome determined for each tumour, resulting in a normalized probe level summarization value and a genotype call for each individual probe58 (link). Circular binary segmentation was performed in R using the DNAcopy BioConductor package. Default parameters included nperm=10,000 (the number of permutations used for Pvalue calculation), α=0.01 (the significance level for the test to accept change points), and random seed=12,345,678 (the seed for the random number generator). This resulted in a segmented file for each tumour sample relative to the corresponding normal sample. Segments containing at least eight markers in which the log2 value was ≥+0.5 or ≤−0.5 were considered regions of gain or loss, respectively.

Genome-wide SNP typing was performed in 13 orexin mutation-positive IH patients, 116 orexin mutation-negative IH patients, and 420 healthy controls using the Genome-Wide Human SNP Array 6.0 according to the manufacturer’s protocols (Affymetrix Inc., Santa Clara, CA). Data generated by the array were analyzed with GeneChip Operating Software and Genotyping Console 4.0 (Affymetrix Inc). We included SNPs that showed genotyping call rates of >97%, MAFs of >5%, and P values of more than the threshold of the Hardy–Weinberg equilibrium (P > 0.001), which was evaluated using the Chi-2 test. We checked unknown familial relationships between subjects in this study with PIHAT values as calculated by PLINK 1.9^{52 (link)}.
When calculating PIHAT values, linkage disequilibrium-based SNP pruning was conducted (r² < 0.5). PCA was performed using the EIGENSTRAT program^{53 (link)} and PLINK 1.9 to evaluate population stratification or differences in genetic background among the study subjects genotyped with the SNP array. In the PCA, 45 JPT (Japanese in Tokyo, Japan), 90 CEU (Utah residents with Northern and Western European ancestry from the CEPH collection), 90 YRI (Nigeria Yoruba in Ibadan, Nigeria), and 45 CHB (Han Chinese in Beijing, China) were utilized that were derived from the International HapMap project.

Genomic DNAs from gastric tumours and matched normal gastric tissues were hybridized on Affymetrix SNP6.0 arrays (Affymetrix, Santa Clara, CA). Data in CEL format was processed in the following order: (1) Normalization: Raw.CEL files were processed using Affymetrix Genotyping Console 4.2. Reference models were created from SNP6.0 profiles of normal gastric tissues according to the hybridization batch. Copy number changes in cell lines and primary tumour samples were determined by using the reference model from primary normal samples. (2) Segmentation: Copy number segmentation data were produced using the circular binary segmentation (CBS) algorithm implemented in the DNAcopy R package. The P-value cutoff for detecting a change-point was 0.01, with a permutation number of 10,000. Copy number gain and loss regions were defined for showing average log ratios of >0.6 and<−1.0, respectively. Illumina HumanMethylation450 (HM450) Infinium DNA methylation arrays were also used to assay DNA methylation levels. Methylation β-values were calculated and background corrected using the methylumi R BioConductor package. Normalization was performed using the BMIQ method (watermelon package in R).