Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing

Abstract

Short-tandem repeat (STR) expansions are an important class of pathogenic genetic variants. Over forty neurological and neuromuscular diseases are caused by STR expansions, with 37 different genes implicated to date. Here we describe the use of programmable targeted long-read sequencing with Oxford Nanopore's ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single, simple assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of expanded and non-expanded STR sites. In doing so, the assay correctly diagnoses all individuals in a cohort of patients (n = 27) with various neurogenetic diseases, including Huntington's disease, fragile X syndrome, cerebellar ataxia (CANVAS) and others. Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing, and identifies non-canonical STR motif conformations and internal sequence interruptions. Even in our relatively small cohort, we observe a wide diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of STR expansion disorders. Finally, we show how the flexible inclusion of pharmacogenomics (PGx) genes as secondary ReadUntil targets can identify clinically actionable PGx genotypes to further inform patient care, at no extra cost. Our study addresses the need for improved techniques for genetic diagnosis of STR expansion disorders and illustrates the broad utility of programmable long-read sequencing for clinical genomics.

Long read sequencing overcomes challenges in the diagnosis of SORD neuropathy

“…3 This report highlights that long-read sequencing methods are capable of correctly phasing biallelic SORD mutations and differentiating SORD from the SORD2P pseudogene, thus addressing two previously discussed difficulties in SORD neuropathy diagnostics. Whilst we applied wholegenome ONT sequencing here, targeted ONT sequencing which sequences a subset of the genome is also feasible 17 and represents an attractive method for more cost-effective analysis of SORD and other relevant genes in CMT. However, as with declining costs of short read NGS, it is likely that the cost of whole genome long read sequencing ($1300 USD at Garvan Institute, Australia) will decrease and result in greater accessibility for clinicians and researchers.…”

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing

“…Further improvements are therefore required before the multiplexing protocol is suitable, for example by combining the Cas9 protocol with the recently introduced "Read Until" feature of ONT sequencing to exclude the background noise. This allows selective sequencing of pre-defined target DNA molecules and has been used to characterize pathogenic repeats (Stevanovski et al 2021). Alternatively, costs could be optimized by analyzing single samples using Flongles, which produce less sequencing data than regular ONT flow cells but reduce costs by 90%.…”

Single Assay Tests for More Than 50 Genetic Disorders

“…Now, researchers from the Garvan Institute of Medical Research in Sydney, Australia, and an international team of collaborators have developed a genetic test that can identify a wide range of hard-to-diagnose neurological and neuromuscular genetic diseases faster and more accurately than existing methods (Stevanovski et al, 2022). The 50 neurological and neuromuscular diseases within this group fall into the category of short tandem repeat (STR) expansions, with 37 different genes implicated to date.…”