Whole-Genome Sequencing and Variant Analysis

Whole-genome sequencing was performed at the University of Washington Center for Mendelian Genomics (University of Washington, Seattle). Initial quality control (QC) entailed DNA quantification, gender validation assay, and molecular fingerprinting with a 63-SNP OpenArray assay derived from a custom exome SNP set. Following successful QC, at least 750 ng of genomic DNA was subjected to a series library construction steps utilizing the KAPA Hyper Prep kit (Roche), automated on the Perkin Elmer Janus platform. Libraries were validated using the Bio-Rad CFX384 Real-Time System and KAPA Library Quantification kit (Roche). Samples were sequenced on a HiSeq X using Illumina’s HiSeq X Ten Reagent Kit (v2.5) to an average depth of 30X. Burrows-Wheeler Aligner^{52 (link)}, Genome Analysis ToolKit^{53 (link)} and SeattleSeq Annotation server build 138 (https://snp.gs.washington.edu/SeattleSeqAnnotation138/) were used to generate BAM, vcf and annotation files, respectively. Homozygosity mapping was performed with PLINK v1.07 software^{54 (link)} using the genotypes generated by the 63-SNP OpenArray assay. Structural variants were called using Lumpy^{55 (link)}. Alignments were visualized using the Integrative Genomics Viewer tool^{56 (link)}. The LMBR1 deletion and ZRS variants were validated by PCR-Sanger sequencing (primers provided in Supplementary Table 3).