title: Whole Exome Sequencing of Homo Sapiens: Male blood
design: Genomic DNA (gDNA) from whole blood was isolated for genetic analysis using the Omega Genomic DNA purification Kit. The coding and flanking intronic regions of gDNA were enriched using the Ampiseq technology as designed and supplied by ThermoFisher Scientific (Ion AmpliSeq Library Kit Plus). A custom Ampliseq panel was designed using the Ion Ampliseq Designer V.3.0 (LifeTechnologies, Carlsbad, CA, USA) that included the COL4A3/A4/A5 genes. This design consisted of 260 primer-pairs that amplify and cover all exons at 99.00%. NGS library construction involved amplicon generation through PCR to select exonic regions, which then were partially digested to ligate adapters and barcodes. Amplicon sizes ranged from 125175bp. Each library was purified and quantified to ensure the highest quality of output sequences. Sequence template preparation, and chip loading was performed by an automated prep system, the IonChef (ThermoFisher Scientific) and sequencing was performed on an Ion GeneStudio-S5 Plus System. Signal detection, base calling and BAM file generation was performed by the Torrent Suite VM built into the Ion GeneStudio-S5  Plus. Sequence reads of each sample were mapped to the human reference genome (hg19) using the Torrent Mapping Alignment Program (TMAP). BAM analysis, variant calling, and annotation were performed by the IonReporter sequencing analysis server, and VCF files were generated for further analysis. Only variants (single nucleotide variant (SNVs)/small indels) with a minor allele frequency (MAF) of 0.03 within the coding regions and their adjacent intronic regions were evaluated. Variants were annotated and classified with respect to their possible impact on protein function, based on the available data from databases and algorithms (Varsome, Franklin-by-Genoox, ClinVar, Mutation Taster, REVEL, SIFT, PolyPhen). Variant classification was based on the American College of Medical Genetics and Genomics (ACMG) guidelines. The presence of variants in the human genome mutation database (HGMD) was also explored. The clinically relevant variant was validated by dye-terminator Sanger DNA re-sequencing performed using the ABI BigDye Terminator v1.1 Cycle Sequencing Kit and the ABI PRISM 3130xl genetic analyzer using appropriate primers.