Genome viewer

Illumina HumanOmni 2.5 M SNP array was used for whole genome SNP genotyping. A total of 200 ng genomic DNA from four members (III:3, III:4, III:5, IV:2) of a family was used as a starting material. Briefly, 0.1 N NaOH was used for DNA denaturation and whole genome was amplified out with Random Primers Mix (RPM) using Multi Sample Master Mix (MSM). Enzymatic fragmentation of the amplified DNA was carried out using Fragmentation Mix (FMS) followed by precipitation using Precipitation Mix 1 (PM1) and 2-propanol. Fragmented DNA was hybridized to BeadChip by denaturing the sample and dispensing 35 ul of the sample onto the BeadChip section followed by incubation for 18 hours at 48 °C in the hybridization oven. Single base extension on BeadChips was performed after washing and staining. Single base extension reaction incorporates labeled nucleotides into the extended primers. Scanning of BeadChips was performed in Illumina iScan using iScan control software. Illumina GenomeStudio software and HomozygosityMapper were used to call loss of heterozygous (LOH) regions while the Illumina Genome Viewer incorporated in GenomeStudio was used to detect copy number variations (CNVs) in the genome. Identity-by-descent (IBD) analysis was carried out using PLINK [26 (link)] to identify shared genomic regions.

CNV analysis was conducted for seven samples in the family pedigree (Fig. 1A). The Illumina cnvPartition v3.2.0 CNV Analysis Plug-in for GenomeStudio Software was used to calculate CNV and confidence values for all chromosomes. The default value was adopted for each parameter of the plug-in. The CNV regions were listed using the Illumina CNV Region Report Plug-in v2.1.1. The distributions of the CNV value, log R ratio, and B allele frequency were visualized using the Illumina Genome Viewer embedded in GenomeStudio Software to confirm whether the detected CNVs in the genes with high LOD scores were false positives or true negatives.