Implementation of Nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinic

Abstract

Background Diagnosis of rare genetic diseases can be a long, expensive and complex process, involving an array of tests in the hope of obtaining an actionable result. Long-read sequencing platforms offer the opportunity to make definitive molecular diagnoses using a single assay capable of detecting variants, characterizing methylation patterns, resolving complex rearrangements, and assigning findings to long-range haplotypes. Here, we demonstrate the clinical utility of Nanopore long-read sequencing by validating a confirmatory test for copy number variants (CNVs) in neurodevelopmental disorders and illustrate the broader applications of this platform to assess genomic features with significant clinical implications. Methods We used adaptive sampling on the Oxford Nanopore platform to sequence 25 genomic DNA samples and 5 blood samples collected from patients with known or false-positive copy number changes originally detected using short-read sequencing. Across the 30 samples (a total of 50 with replicates), we assayed 35 known unique CNVs (a total of 55 with replicates) and one false-positive CNV, ranging in size from 40 kb to 155 Mb, and assessed the presence or absence of suspected CNVs using normalized read depth. Results Across 50 samples (including replicates) sequenced on individual MinION flow cells, we achieved an average on-target mean depth of 9.5X and an average on-target read length of 4805 bp. Using a custom read depth-based analysis, we successfully confirmed the presence of all 55 known CNVs (including replicates) and the absence of one false-positive CNV. Using the same CNV-targeted data, we compared genotypes of single nucleotide variant loci to verify that no sample mix-ups occurred between assays. For one case, we also used methylation detection and phasing to investigate the parental origin of a 15q11.2-q13 duplication with implications for clinical prognosis. Conclusions We present an assay that efficiently targets genomic regions to confirm clinically relevant CNVs with a concordance rate of 100%. Furthermore, we demonstrate how integration of genotype, methylation, and phasing data from the Nanopore sequencing platform can potentially simplify and shorten the diagnostic odyssey.

NanoImprint: A DNA methylation tool for clinical interpretation and diagnosis of common imprinting disorders using nanopore long‐read sequencing

“…Full reports can be found in the Supporting Information. disturbances can afterwards be delineated from the sequencing data using copy number-, homozygosity-and structural variant analysis as shown by others (Cuenca-Guardiola et al, 2023;Greer et al, 2023;Yamada et al, 2023).…”

Nanopore adaptive sampling to identify the NLR-gene family in melon (Cucumis meloL.)

“…NAS requires a standard library preparation, eliminates the need for DNA amplification, circumvents laborious or expensive experimental design or probes synthesis, and offers real-time selective enrichment (Martin et al, 2022; Miyatake et al, 2022). NAS has been used in clinical settings and for the enrichment of metagenomic samples (Kipp et al, 2021; De Meulenaere et al, 2022; Martin et al, 2022; Greer et al, 2023; Hewel et al, 2023; Su et al, 2023; Wrenn & Drown, 2023). Therefore, NAS emerges as a promising approach for studying target regions, especially those that are highly complex, such as disease-associated repeat loci in humans (Miyatake et al, 2022; Stevanovski et al, 2022).…”

Editorial: Novel applications of ONT technologies in genomics and transcriptomics

“…This Research Topic begins with a comprehensive review “Resolving complex structural variants via nanopore sequencing” by Romagnoli et al which discussed the successful applications of ONT approach for recent developments of NGS (next-generation sequencing) platforms for detection of structural variants (SVs) as the hallmark of genome instability, potentially leading to pathologic conditions. ONT long-reads and its combination with the short reads have already been proven to be invaluable in overcoming limitations of short-read sequencing to resolve wide and structurally complex SVs ( Mahmoud et al, 2019 ; Cleal and Baird, 2022 ; Greer et al, 2023 ). This review also discusses bioinformatics methods that improve the identification of SVs associated with human pathological conditions, discussing the possibility of introducing nanopore sequencing technology into routine clinical diagnostics.…”

“…with the short reads have already been proven to be invaluable in overcoming limitations of short-read sequencing to resolve wide and structurally complex SVs (Mahmoud et al, 2019;Cleal and Baird, 2022;Greer et al, 2023). This review also discusses bioinformatics methods that improve the identification of SVs associated with human pathological conditions, discussing the possibility of introducing nanopore sequencing technology into routine clinical diagnostics.…”