Whole Exome Sequencing for Variant Identification

Blood samples of the probands and their parents were collected for identifying the source of variants. Genomic DNAs were extracted from blood samples using the Qiagen Flexi Gene DNA kit (Qiagen, Hilden, Germany). WES was performed using a NextSeq500 sequencing instrument (Illumina, San Diego, California, United States) following previously described standard procedures (Wang et al., 2018 (link); Luo et al., 2022 (link)). The sequencing data were generated by massively parallel sequencing with an average depth of >125x and >98% coverage of the capture region on the chip for obtaining high-quality reads that were mapped to the Genome Reference Consortium Human genome build 37 by Burrows-Wheeler alignment. Single-nucleotide point variants and indels were called with the Genome Analysis Toolkit.