Iscan control software

BeadChip Arrays were scanned with HiScan Array Scanner (Illumina) using the iScan Control Software (Illumina). Genes and probes transcript levels were obtained from Illumina Intensity Data (.idat) files, applying quantile normalization and background subtraction implemented by the GenomeStudio Gene Expression Module v1.0 Software (Illumina). All experiments in each condition reported were performed on triplicate biological samples. Signals associated with a p-value > 0.05 in all samples were discarded from the analysis. Cut-off for up- and downregulation of gene expression was set to twofold change threshold in all the analyses performed.

The 9827 selected SNPs were submitted to the Illumina Assay Design Tool for design score calculation (www.illumina.com) and 8580 of them were successfully synthesized by Illumina manufacturing processes. The SNP genotyping assay was performed on an Illumina^® Infinium HD iSelect Custom Genotyping Array according to the standard Illumina's protocol, using 200 ng of genomic DNA per sample. The extension and staining steps were operated on a Tecan Evo‐150 liquid‐handling robot. Fluorescence intensities were read with Illumina^® iScan Control software and allele calling was carried out using the Genome Studio v2.0 software (Illumina Inc., San Diego, CA, USA). All data were visually inspected and manually re‐scored if any errors were evident in the calling of the SNP clusters by the default algorithm. Reproducibility error rates were calculated between the control sample and SNP replicates.

Infinium HD Assay Ultra with Illumina multi-sample DNA Analysis BeadChips was performed to detect copy number variants CNVs (duplications, deletions, loss of heterozygosity) in iPSC clones compared with blood from the same case. The data were imported from iScan Control Software into GenomeStudio 2.0 Genotyping Module Software provided by Illumina for the analysis.

Genomic DNA (gDNA) was extracted from flash-frozen liver tissue with PureLink Genomic DNA Mini Kit (Thermo) and quantified using Nanodrop (Thermo). In total 1 μg of isolated gDNA was bisulfite converted, denatured, fragmented and hybridized to Infinium Methylation Bead Chip, following the manufacturer protocol (Infinium MethylationEPIC kit, Illumina). BeadChips were imaged using an Illumina Scan System and intensity was determined by iScan Control Software (Illumina). Sample intensities were normalized using functional normalization from the minfi package (v1.24.0)^{60 (link)}. Probes failing a detection p-value threshold (0.01) in at least 50% of samples were removed, as were probes identified as containing a SNP with a MAF >0.05. Differentially methylated probes were identified by applying limma (v3.34.3)^{54 (link)} contrasts to M values (absolute change in beta value >0.1, FDR-corrected P-value < 0.05). Differentially methylated regions were identified using DMRcate (v1.14.0)^{61 (link)} setting a threshold of absolute change in beta value in >0.1 and of Stouffer’s value in <0.05.

The Gentra Puregene Buccal Cell Kit (Qiagen, Hilden, Germany) was used to extract DNA from the buccal cell samples. The Human Exome Bead Chip Kit v1.0 and v1.1 Illumina (San Diego, CA, USA), which consists of 243,345 putative functional exonic markers, was utilized with Illumina iScan for the microarray. All DNA samples were processed according to the manufacturer’s protocol, and iScan control software (Illumina) was used to acquire data. DNA extraction and the microarray genotyping and analysis were performed at the genetics research laboratory of the Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. Genotyping was carried out in this lab from 2016 to 2018 using the same chip and procedures.

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.

Jirel DNA samples were genotyped using Illumina's Human660W-Quad v1 BeadChip (Illumina Inc., San Diego, CA, USA) containing ~550,000 SNP loci. A total of 200 ng of genomic DNA for each sample was processed according to Illumina's Infinium HD Assay Ultra protocol. BeadChips were imaged on Illumina's iScan System with iScan Control Software (v.3.2.45). Normalization of raw image intensity data, genotype clustering, and individual sample genotype calls was performed using Illumina's GenomeStudio software (v2010.2), Genotyping Module (v1.7.4). Illumina's predefined genotype cluster boundaries were used to denote SNP genotype cluster positions (Human660W-Quad_v1_H.egt). Genotype assay quality control measures were assessed with Illumina's internal assay performance metrics.