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

Abstract

More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. 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 assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort ( n = 37) including patients with various neurogenetic diseases ( n = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care.

The Evolving Role of Diagnostic Genomics in Kidney Transplantation

“…In the future, advances in these platforms, such as short-read and long-read technology, may be useful for those with previously uninformative genomic sequencing. 49 , 50 , 51 …”

“…In the future, advances in these platforms, such as short-read and long-read technology, may be useful for those with previously uninformative genomic sequencing. [49][50][51] These testing platforms are increasingly becoming a useful tool in diagnosis of kidney disease in the context of panel testing according to clear clinical phenotype. An example of its use is in the case of Alport syndrome, where a patient with the applicable phenotype would only need to be tested for variants in the genes of importance, COL4A3, COL4A4, and COL4A5.…”

Rapid PCR-Based Nanopore Adaptive Sequencing Improves Sensitivity and Timeliness of Viral Clinical Detection and Genome Surveillance

“…Sequencing enrichment of the human genome, tumor genes, or specific species in a mock microbial community has been observed by adaptive sequencing. For human genetic diagnosis, adaptive sequencing has been used to accurately identify pathogenic structural variants ( Miller et al, 2021 ; Stevanovski et al, 2022 ) and determine the structure of the small supernumerary marker chromosomes ( Mariya et al, 2022 ). Combined with the enzymatic removal of the host background, adaptive sequencing obtained a 113.41-fold enrichment of microbial DNA in respiratory tract samples ( Gan et al, 2021 ).…”

Detection of repeat expansions in large next generation DNA and RNA sequencing data without alignment

“…The development of long-read sequencing (LRS) has recently been shown to offer immense utility in analysis of STR loci 18 . LRS is particularly useful in uncovering the architecture and sizing of large and complex REs such as those associated with cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS) 19 or benign adult familial myoclonic epilepsy (BAFME) 20 , but the relative expense and high volumes of data associated with obtaining equivalent sequencing depth across the genome offer significant barriers to its use in large-scale studies.…”