2.2. Genetic Testing

Whole-exome sequencing (WES) was performed by WuXi NextCODE Genomics, Shanghai, China (CLIA Lab ID: 99D2064856). Briefly, exome capture was performed using the Agilent SureSelect Human All Exon V5, Illumina TruSeq Rapid PE Cluster, and SBS kits (Agilent Technologies, Santa Clara, CA, USA). Illumina HiSeq provided the 2000/2500 platform for WES. Read alignment was referenced to the human genome sequence (http://genome.ucsc.edu, accessed on 7 July 2000) using the Burrows-Wheeler Aligner v.0.6.2. Duplicate paired-end reads were marked with Picard v.1.55 (https://broadinstitute.github.io/picard/, accessed on 5 September 2020). The base quality score recalibration, indel realignment, and variant discovery were performed using the Genome Analysis Toolkit v.2.3–9. Variants were annotated using a pipeline developed in-house and filtered in the Genome Aggregation Database (gnomAD), Exome Variant Server, dbSNP databases, and Exome Aggregation Consortium. All putative pathogenic variants observed by ES were validated by Sanger sequencing (primer sequences and amplification protocols are available upon request).