WES of Homo sapiens: 4 years old male blood
We employed a Trio-WES approach to identify causal variants within family trios. In brief, genomic DNA was extracted and enriched using a Qiagen DNA Blood Midi/Mini kit (Qiagen GmbH, Hilden, Germany) following standard protocols. Subsequently, 50 nanograms of DNA were fragmented to approximately 200 base pairs using fragmentation enzymes. The resulting DNA fragments were end-repaired and an A base was added to the 3' end. Following this, the DNA fragments were ligated with barcoded sequencing adaptors, and fragments of approximately 320 base pairs were collected using XP beads.After PCR amplification, the DNA fragments were hybridized and captured by NanoWES, in accordance with the manufacturers protocol. The hybrid products were eluted, collected, and subjected to further PCR amplification and purification. The libraries were then quantified using qPCR, and size distribution was determined using NanoWES (Berry Genomics, China). The genomic sequencing was conducted using the Novaseq6000 platform (Illumina, San Diego, USA) in 150 bp pair-end sequencing mode. Raw image files were processed using CASAVA v1.82 for base calling and generating raw data.Alignment of the sequencing reads to the human reference genome (hg19/GRCh37) was performed using the BurrowsWheeler Aligner tool, with PCR duplicates subsequently removed using Picard v1.57 (http://picard.sourceforge.net/). Variant calling was conducted using the Verita Trekker Variants Detection System by Berry Genomics and the third-party software GATK (https://software.broadinstitute.org/gatk/). Variant annotation and interpretation were carried out using ANNOVAR [14] and the Enliven Variants Annotation Interpretation System authorized by Berry Genomics. Annotation databases primarily included human population databases such as gnomAD, the 1000 Genome Project, Berrybig data population database, dbSNP, among others; in silico prediction algorithms including SIFT, FATHMM, MutationAssessor, CADD, SPIDEX; and disease and phenotype databases such as OMIM, ClinVar, HGMD, HPO.