Whole-exome Sequencing Protocol for Variant Analysis

Whole-exome sequencing was performed on genomic DNA from patients and donors using QIAmp DNA Micro Kit (Qiagen). For whole-exome capture libraries, Agilent Human All Exon V7 baits were used. Samples were sequenced on Illumina NovaSeq 6000 (PE150) with mean coverage of 50× to generate 150 bp paired-end reads. After filtering using Trimmomatic v0.33 with default parameters (LEADING:15 TRAILING:15 SLIDINGWINDOW:4:15 MINLEN:50), reads were mapped against the GRCh38 reference genome using Burrows-Wheeler Aligner 3 (BWA-mem v0.7.17).³¹ (link) Duplicate reads were removed using Picard Tools (http://broadinstitute.github.io/picard/). Genome Analysis Toolkit 7³² (link) (GATK v4.2.4) was used for base quality recalibration and variant calling. The resulting variant call format (VCF) files for each sample were combined and genotyped using GATK CombineGVCFs and GenotypeGVCFs, respectively. For 15 of 18 SNPs with insufficient coverage, genotyping was performed using KASPar assays (LGC Biosearch Technologies).