WES-Seq of Homo sapiens, Keratoconus Patient: Sample KC238
The genomic DNA (gDNA) was quantified using the Picogreen assay (cat# P7589, ThermoFisher) on VictorX2 multilabel plate reader (Perkin Elmer). DNA integrity was checked using gDNA screen tape (Catalog # 5067-5365, Agilent Technologies) and gDNA reagents (Catalog # 5067-5366, Agilent Technologies) on 4200 Tapestation system (cat. # G2991AA, Agilent Technologies).  1ug of gDNA was used as input material for SureSelect XT Human All Exon V5 library construction.  gDNA fragmentation was conducted using a Covaris model LE220 instrument to target DNA fragment size of 150 to 200bp. T4 DNA Polymerase and T4 PNK from SureSelectXT Reagent kit (cat. # G9611B, Agilent Technologies) were added to the sheared or purified DNA fragments and the mixture was incubated at 20°C for 30minutes for end repair reaction.  Exo(–) Klenow enzyme from SureSelectXT Reagent kit (cat. # G9611B, Agilent Technologies) was used for subsequent dA-tailing and the mixture was incubated at 37°C for 30 minutes. SureSelect XT library adapters were ligated to the dA-tailed DNA fragments and then amplified with SureSelect Primers and Herculase II Fusion DNA Polymerase from the kit on C1000 Touch Thermal cycler (cat#  185-1196, Bio-Rad). Amplified library was validated with D1000 ScreenTape (cat.# 5067-5582, Agilent Technologies) and  D1000 Reagents (cat.# 5067-5583, Agilent Technologies) on 4200 Tapestation system(cat. # G2991AA, Agilent Technologies). The validated library was used for hybridization process with SureSelect XT Human All Exon V5 library kit (cat. # 5190-6208, Agilent Technologies) and final captured library was amplified and validated again with D1000 ScreenTape (cat.# 5067-5582, Agilent Technologies) and  D1000 Reagents (cat.# 5067-5583, Agilent Technologies) on 4200 Tapestation system(cat. # G2991AA, Agilent Technologies) for size checking and picogreen assay (cat# P7589, ThermoFisher) on VictorX2 multilabel plate reader (Perkin Elmer) for quantification. The validated libraries were then normalized to 10nM and loaded onto Hiseq2500 Rapid mode (101x2PE run) after diluting to desired loading concentration (8pM) and sequenced by Psomagen.