![]() ![]() The ability to sequence multiple target loci concurrently, rather than consecutively, has increased diagnostic throughput and helped refine the mutation spectra of hundreds of genetic disorders. The universal adoption of short-read next generation sequencers by both research and diagnostic laboratories has transformed the availability and repertoire of molecular genetic assays. Our findings add to a growing body of literature describing the diagnostic utility of long-read sequencing. We demonstrate a number of technical advantages over existing wet-laboratory approaches, including in silico size selection of a mixed pool of amplification products, and the relative ease with which an automated informatics workflow can be established. Pairwise comparison between these data and the previously determined benchmark alleles revealed 100% identity of the variant-containing sequences. Long-read nanopore sequencing was then performed on locus-specific amplicons. To validate the accuracy of the long-read technology, we first used Sanger sequencing to confirm the integration sites and derive curated benchmark sequences of the variant-containing alleles. Here, we describe three diagnostic cases in which pathogenic mobile element insertions were refractory to characterization by short-read sequencing. “Third-generation” long-read sequencers are increasingly being deployed as an orthogonal adjunct technology, but their full potential for molecular genetic diagnosis has yet to be exploited. Inherent limitations of short-read technology, notably for the detection and characterization of complex insertion-containing variants, are offset by the ability to concurrently screen many disease genes. Short-read next generation sequencing (NGS) has become the predominant first-line technique used to diagnose patients with rare genetic conditions. ![]()
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