Targeted long-read sequencing enriches disease-relevant genomic regions of interest to provide complete Mendelian disease diagnostics
Kenji Nakamichi, … , Russell N. Van Gelder, Debarshi Mustafi
Kenji Nakamichi, … , Russell N. Van Gelder, Debarshi Mustafi
Published September 12, 2024
Citation Information: JCI Insight. 2024;9(20):e183902. https://doi.org/10.1172/jci.insight.183902.
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Research Article Genetics Ophthalmology

Targeted long-read sequencing enriches disease-relevant genomic regions of interest to provide complete Mendelian disease diagnostics

Abstract

Despite advances in sequencing technologies, a molecular diagnosis remains elusive in many patients with Mendelian disease. Current short-read clinical sequencing approaches cannot provide chromosomal phase information or epigenetic information without further sample processing, which is not routinely done and can result in an incomplete molecular diagnosis in patients. The ability to provide phased genetic and epigenetic information from a single sequencing run would improve the diagnostic rate of Mendelian conditions. Here, we describe targeted long-read sequencing of Mendelian disease genes (TaLon-SeqMD) using a real-time adaptive sequencing approach. Optimization of bioinformatic targeting enabled selective enrichment of multiple disease-causing regions of the human genome. Haplotype-resolved variant calling and simultaneous resolution of epigenetic base modification could be achieved in a single sequencing run. The TaLon-SeqMD approach was validated in a cohort of 18 individuals with previous genetic testing targeting 373 inherited retinal disease (IRD) genes, yielding the complete molecular diagnosis in each case. This approach was then applied in 2 IRD cases with inconclusive testing, which uncovered noncoding and structural variants that were difficult to characterize by standard short-read sequencing. Overall, these results demonstrate TaLon-SeqMD as an approach to provide rapid phased-variant calling to provide the molecular basis of Mendelian diseases.

Authors

Kenji Nakamichi, Jennifer Huey, Riccardo Sangermano, Emily M. Place, Kinga M. Bujakowska, Molly Marra, Lesley A. Everett, Paul Yang, Jennifer R. Chao, Russell N. Van Gelder, Debarshi Mustafi

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Figure 4

Haplotype-resolved assembly of complex phased variants provides insight into differential disease phenotypes.

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Haplotype-resolved assembly of complex phased variants provides insight ...
(A) Two affected siblings with USH2A-associated Usher syndrome were found to have 3 pathogenic variants in USH2A from clinical testing without phase information. Targeted long-read sequencing demonstrated that the unaffected father harbored 2 pathogenic variants in cis, whereas the unaffected mother harbored 1 pathogenic variant. Targeted long-read GS correctly identified the variant architecture from the probands alone with the 2 variants inherited from the father in trans to the variant inherited from the mother. Variant architecture can influence disease progression, as evidenced in ABCA4-associated Stargardt disease. (B) An adolescent subject with severe disease, as exhibited by chorioretinal atrophy with a large region of hypoautofluorescence in the central macula, had 3 pathogenic alleles, 2 of which were severe and were in a trans configuration. (C) In comparison, a middle-aged individual with mild disease, as exhibited by hyperautofluorescent flecks without atrophy in the central macula, also had 3 pathogenic alleles, but had 2 hypomorphic alleles in cis, both of which were in trans to a severe allele.