Targeted long-read sequencing resolves complex structural variants and identifies missing disease-causing variants

Abstract

BACKGROUND: Despite widespread availability of clinical genetic testing, many individuals with suspected genetic conditions do not have a precise diagnosis. This limits their opportunity to take advantage of state-of-the-art treatments. In such instances, testing sometimes reveals difficult-to-evaluate complex structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in specific genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. METHODS: Targeted long-read sequencing (T-LRS) was performed on 33 individuals using Read Until on the Oxford Nanopore platform. This method allowed us to computationally target up to 100 Mbp of sequence per experiment, resulting in an average of 20x coverage of target regions, a 500% increase over background. We analyzed patient DNA for pathogenic substitutions, structural variants, and methylation differences using a single data source. RESULTS: The effectiveness of T-LRS was validated by detecting all genomic aberrations, including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences, previously identified by prior clinical testing. In 6/7 individuals who had complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, which led, in one case, to a change in clinical management. In nine individuals with suspected Mendelian conditions who lacked a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in five and variants of uncertain significance in two others. CONCLUSIONS: T-LRS can accurately predict pathogenic copy number variants and triplet repeat expansions, resolve complex rearrangements, and identify single-nucleotide variants not detected by other technologies, including short-read sequencing. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority candidate genes and regions or to further evaluate complex clinical testing results. The application of T-LRS will likely increase the diagnostic rate of rare disorders.

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing

“…The enrichment of targets in human cells (26)(27)(28)(29)(30) or species in mock communities (27,31) or fecal samples of lions ( 32) was previously demonstrated. In the present study, we used clinical metagenomic samples obtained from vaginal swabs of pregnant women to evaluate the performance to deplete the high content of human DNA that heavily impairs downstream microbiome analyses.…”

Compartmap enables inference of higher-order chromatin structure in individual cells from scRNA-seq and scATAC-seq

“…Recent work has shown that rare SVs are abundant in human populations 86 , and can be missed even by long-read assembly-based approaches 87 . With improved prioritization schemes, the clinical benefit of long-read sequencing can be maximized relative to its cost 88 . We believe compartmap can serve a key role in facilitating this process for novel SVs, maximizing the value of both existing and new data, thereby accelerating discoveries.…”

A guide for the diagnosis of rare and undiagnosed disease: beyond the exome

“…With continuing improvements, long-read technologies can enable sequencing of more such difficult-to-sequence regions, identifying new associations between genomic regions and genetic disorders. Several groups have exemplified the clinical significance of targeted long-read sequencing using CRISPR/Cas9 [114] mediated methods [115,116] or computational adaptive sampling [117] for enrichment of specific regions of the genome. In a small cohort of 22 patients with known canonical and complex SVs, Miller et al [117] demonstrated that targeted LRS can not only detect all SVs previously identified with clinical testing (n = 46) but also discovered variants (n = 41) that were missed by the clinical test.…”

“…Several groups have exemplified the clinical significance of targeted long-read sequencing using CRISPR/Cas9 [114] mediated methods [115,116] or computational adaptive sampling [117] for enrichment of specific regions of the genome. In a small cohort of 22 patients with known canonical and complex SVs, Miller et al [117] demonstrated that targeted LRS can not only detect all SVs previously identified with clinical testing (n = 46) but also discovered variants (n = 41) that were missed by the clinical test. Targeted long-read sequencing has the potential to be used clinically for patients with suspected complex SVs and tandem repeats in candidate genes.…”