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.

Keywords: Adaptive sampling; Clinical testing; Copy number variants; Genome analysis; Long-read sequencing; Neurodevelopmental disorders; Oxford Nanopore Technologies; Targeted sequencing.

Fig. 1

Overview of the Oxford Nanopore Technologies (ONT) copy number variant (CNV) confirmation assay. CNV coordinates are initially detected with short-read sequencing and then targeted and confirmed using ONT adaptive sampling along with a custom bioinformatics pipeline

Fig. 2

Examples of copy number variants (CNVs) identified by our pipeline. A Scatter plot of the regional mean depth for a Coriell sample (NA04099/SM4716) known to carry a deletion. A point was plotted for each of the five control regions, the two pad regions, and the deleted region. Each plotted point denotes the mean depth of the region normalized by either the mean depth across all control regions (left side of plot) or the mean depth across all control and pad regions (right side of plot). For each normalization approach, dashed lines are plotted to indicate three standard deviations from the mean of the normalized control regions (gray) and the normalized control and pad regions (green). B Read depth plot for a Coriell sample (NA04099/SM4716) known to carry a deletion. The mean depth in the deleted region was normalized by the mean depth across all control regions, calculated in non-overlapping windows that were 1% of the target size. The red dashed line indicates the mean depth ratio across the deleted region, and the green dashed lines indicate the mean depth ratios of the pad regions directly adjacent to the deleted region. C Scatter plot of the regional mean depth for a blood sample (Individual_5/SM7419) from a healthy individual known to carry a duplication. D Read depth plot for a blood sample (Individual_5/SM7419) from a healthy individual known to carry a duplication

Fig. 3

Cutoff thresholds for copy number variant (CNV) confirmation. A Boxplot displays of the cutoffs used to confirm deletions and duplications across 50 samples and 56 CNVs (including the false-positive CNV). The cutoff thresholds for CNVs were set at three standard deviations from the mean of either the mean depth ratios of the five control regions or the mean depth ratios of the five control regions and two pad regions, whichever was larger or smaller for deletions and duplications, respectively. ^***, p < 0.001. The dashed gray line denotes a mean depth ratio of 1, which would be the expected mean depth ratio for a region not affected by a CNV. B Scatter plot of the difference between the CNV mean depth ratio and its dynamically determined cutoff for each of the 56 deletion, duplication, or control variants. The difference for deletions was calculated as the CNV depth ratio subtracted from the deletion depth ratio cutoff, and the difference for duplications was calculated as the duplication depth ratio cutoff subtracted from the CNV depth ratio, such that confirmed CNVs appear above zero, denoted by the red dashed line

Fig. 4

Scatter plot of the mean copy number variant (CNV) depth ratios for 56 CNVs (including the false-positive CNV), with differing expected copy numbers, across a range of mean control depths. The dashed horizontal lines indicate the expected mean depth ratio for each expected copy number

Fig. 5

Heatmap of the proportion of four control regions that matched between Coriell samples sequenced previously with short-read sequencing and in the current study using long-read sequencing with adaptive sampling. The regions are matched based on single nucleotide variant content. The analysis also includes samples sequenced multiple times with long-read sequencing and short-read sequencing, as well as a sample sequenced using only short reads and thus with no long-read sequencing match

Fig. 6

Integrative Genomics Viewer (IGV) snapshot of the SNRPN promoter CpG island region in Coriell sample NA22397, known to have a duplication of chromosome 15q. Reads assigned to haplotype 1 (Hap 1) are methylated (denoted by red) while reads assigned to haplotype 2 (Hap 2) are unmethylated (denoted by blue)