DNA extraction and full genome analysis pipeline
Fresh peripheral blood samples were collected from the patient at age 4 and her two biological parents. After removing the red blood cells by RBC lysis buffer (Qiagen), the high-molecular-weight genomic DNA (HMW gDNA) was extracted from the white blood cells (WBCs) of each sample by a plug lysis process with the Bionano Prep™ kit (Bionano Genomics). HMW gDNA from the trio then underwent 10x Genomics (10xG) Linked‐Reads Chromium whole genome sequencing (WGS) on the NovaSeq 6000 Sequencing System (Illumina) to 40-60X read depth and optical mapping on the Bionano Genomics Saphyr System to 60X coverage. The FASTQ files were processed and aligned with GRCh38 (hg38, Genome Reference Consortium) via Long Ranger BASIC and ALIGN Pipelines (10x Genomics) for linked-read alignment, variant calling, phasing, and structural variant Calling. Thede novo , phased genome assemblies of the patient and her two biological parents were compared to identify single nucleotide variants (SNVs) and structural variants (SVs). After filtering out synonymous SNVs and common SNVs/SVs based on data from the 1000 Genomes Project, Exome Aggregation Consortium (ExAC), and Genome Aggregation Database (gnomAD), the genes affected by SNVs, SVs, or a combination of SNVs and SVs were examined in detail. In consultation with the clinicians, a thorough literature search was done on candidate genes with variants (both SNVs and SVs) predicted as deleterious by multiple tools in the web-based wANNOVAR suite to look for genotype-phenotype correlation. All candidate variants were confirmed by Sanger sequencing of PCR products amplified from the individuals. The following primers were used for theBCAP31 mutation: forward 5’-AGCGAACGGAAGTTTGTG‐3’ and reverse 5’‐GGTGGGAGGGCATCATTT‐3’.