Oligonucleotide baits

Exome sequencing was performed on genomic DNA extracted from blood. Quartet exome sequencing in family 1 was performed through the NIH Intramural Sequencing Center (NISC) using the Illumina (San Diego, CA) TruSeq Exome Enrichment Kit and Illumina HiSeq 2500 sequencing instruments. Variants were analyzed using Seqr (Center for Mendelian Genomics) and searched for in dbSNP, NHLBI EVS, Exome Aggregation Consortium (ExAC Browser and GEM.app).8For patient 2, all coding exons (exons 2–66) and the 10–20 bases flanking intronic sequences of COL12A1 (NM_004370.5) were captured with oligonucleotide baits (Agilent Technologies, Santa Clara, CA; Roche, Pleasanton, CA; IDT, Coralville, IA). Next‐generation sequencing (NGS) was performed on Illumina platforms (San Diego, CA), and quality control standards were implemented to achieve a minimum of 50× and an average of 350× depth‐of‐sequence read coverage. Sequence and copy number variant (CNV) analyses were performed with validated NGS methods, allowing for concurrent analysis of sequence variants and exonic CNVs. Diagnostic findings were confirmed by alternate methods.9 Patient 3 and 4 underwent diagnostic clinical whole‐exome sequencing via a CLIA‐certified lab (GeneDx).

Each gene was targeted with oligonucleotide baits (Agilent Technologies, Santa Clara, CA; Roche, Pleasanton, CA; IDT, Coralville, IA) to capture exons, the 10 to 20 bases flanking intronic sequences, and noncoding regions of clinical interest. Baits were iteratively balanced to obtain a minimum of 50X and an average of 350X depth-of-sequence read coverage across all targeted areas. Sequencing was performed on HiSeq and NovaSeq instruments (Illumina, San Diego, CA). A suite of bioinformatics methods was used to identify single nucleotide variants, small and large insertions/deletions, exon-level deletions and duplications, and rare structural or mosaic variants [20 , 21 ].
Genomic DNA extracted from patient blood or saliva was processed by next-generation sequencing as described previously [20 ]. Variants requiring confirmation were confirmed using an orthogonal method, such as PacBio sequencing (Pacific Biosciences, Menlo Park, CA) or exon-focused microarray-based comparative genomic hybridization (Agilent Technologies, Santa Clara, CA) [21 ]. Clinically reported variants and de-identified clinical information, if provided, were collected for analyses.