Long-read sequencing for molecular diagnostics in constitutional genetic disorders.

Long-read sequencing (LRS) has been around for more than a decade, but widespread adoption of the technology has been slow due to the perceived high error rates and high sequencing cost. This is changing due to the recent advancements to produce highly accurate sequences and the reducing costs. LRS promises significant improvement over short read sequencing in four major areas: (1) better detection of structural variation (2) better resolution of highly repetitive or nonunique regions (3) accurate long-range haplotype phasing and (4) the detection of base modifications natively from the sequencing data. Several successful applications of LRS have demonstrated its ability to resolve molecular diagnoses where short-read sequencing fails to identify a cause. However, the argument for increased diagnostic yield from LRS remains to be validated. Larger cohort studies may be required to establish the realistic boundaries of LRS's clinical utility and analytical validity, as well as the development of standards for clinical applications. We discuss the limitations of the current standard of care, and contrast with the applications and advantages of two major LRS platforms, PacBio and Oxford Nanopore, for molecular diagnostics of constitutional disorders, and present a critical argument about the potential of LRS in diagnostic settings.

Genome sequencing increases diagnostic yield in clinically diagnosed Alagille syndrome patients with previously negative test results.

PURPOSE: Detection of all major classes of genomic variants in a single test would decrease cost and increase the efficiency of genomic diagnostics. Genome sequencing (GS) has the potential to provide this level of comprehensive detection. We sought to demonstrate the utility of GS in the molecular diagnosis of 18 patients with clinically defined Alagille syndrome (ALGS), who had a negative or inconclusive result by standard-of-care testing. METHODS: We performed GS on 16 pathogenic variant-negative probands and two probands with inconclusive results (of 406 ALGS probands) and analyzed the data for sequence, copy-number, and structural variants in JAG1 and NOTCH2. RESULTS: GS identified four novel pathogenic alterations including a copy-neutral inversion, a partial deletion, and a promoter variant in JAG1, and a partial NOTCH2 deletion, for an additional diagnostic yield of 0.9%. Furthermore, GS resolved two complex rearrangements, resulting in identification of a pathogenic variant in 97.5% (n = 396/406) of patients after GS. CONCLUSION: GS provided an increased diagnostic yield for individuals with clinically defined ALGS who had prior negative or incomplete genetic testing by other methods. Our results show that GS can detect all major classes of variants and has potential to become a single first-tier diagnostic test for Mendelian disorders.