Genomic Yield of Long-Read Sequencing in Congenital Heart Disease

BACKGROUND: Congenital heart disease (CHD) is the most common birth defect. Ninety percent of isolated cases remain genotype-elusive despite short-read genome sequencing (GS). Our goal was to assess genomic yield with long-read GS compared with short-read GS in CHD. METHODS: We performed Illumina short-read GS on 1101 CHD probands. In a subset of 46 genotype-elusive probands, we performed additional PacBio long-read GS as well as parental sequencing of 7 complete trios. We compared variant calls genome-wide, including across dark and CHD genes in samples with paired short-read and long-read GS. RESULTS: Paired analysis of the 46 probands revealed that genome-wide, long-read GS had 1.01- to 7.93-fold higher call rates of single nucleotide variants, deletions, duplications, and insertions but fewer indels and inversions compared with short-read GS. With a genome-wide sequencing depth of 18.3×, long-read GS had higher coverage for 10 Tier 1 CHD genes and dark genes, but nonuniform and low sequencing depth (<10×) in intragenic regions of 4 of these genes. Long-read GS was better able to resolve complex structural variants and the size of large repeat expansions in 59 known disease-causing regions. For example, long-read GS accurately characterized a complex de novo structural variant upstream of ZEB2 in a CHD proband who had an extracardiac phenotype overlapping with Mowat-Wilson syndrome. CONCLUSIONS: Long-read GS demonstrated higher genome-wide variant call yield compared with short-read GS, better coverage of several cardiac-relevant genes, and better resolution of complex structural variants, including tandem repeat expansions. Long-read GS may provide an option for a subset of patients with CHD who remain genotype-elusive on short-read GS.