Long-range PCR-based NGS applications to diagnose Mendelian retinal diseases

Purpose: To develop a flexible, cost-efficient next-generation sequencing (NGS) protocol for genetic testing. Methods: Long-range polymerase chain reaction (LR PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n=35) of loci associated with retinal diseases (RDs). Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with RD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, (C) 25 undiagnosed after exome sequencing (ES). Results: The method was validated with 100% sensitivity on cohort A. LR PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 CNVs could be characterized. LR PCR libraries spike-in extended coverage data of ES. Read phasing confirmed compound heterozygosity in 5 probands. Conclusion: The proposed sequencing protocol provided deep coverage of the entire gene including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, ii) to elucidate missing heritability cases, iii) to characterize breakpoints of CNVs at the nucleotide level, iv) to extend WES data to non-coding regions by spiking-in LR libraries, and v) to help with phasing of candidate variants.