Nanopore Targeted Sequencing — Adaptive Sampling, CRISPR-Cas9 Enrichment & Amplicon-Based Targeted Long-Read Sequencing for SNVs, SVs & Epigenetics
CD Genomics provides Nanopore targeted sequencing with three complementary enrichment strategies — adaptive sampling for real-time software-based enrichment, CRISPR-Cas9 (nCATS) for amplification-free modification-preserving capture, and long-range amplicon sequencing for high-depth hotspot screening. Each method preserves the long-read, single-molecule characteristics of the Oxford Nanopore platform, enabling simultaneous detection of SNVs, structural variants, repeat expansions, and DNA base modifications (5mC, 5hmC, 6mA) from enriched target regions.
Targeted sequencing occupies a specific and essential position in the genomics toolkit: it delivers the sequencing depth, cost efficiency, and analytical focus required to detect variants, modifications, and structural rearrangements at specific genomic loci without sequencing the entire genome. For the past decade, targeted sequencing has been synonymous with short-read hybridization capture and PCR-based amplicon panels — methods that are effective for point mutations and small indels in well-characterized genomic regions but systematically fail for structural variants, repetitive elements, GC-rich promoters, tandem repeats, and epigenetically modified bases. These failure modes are not incidental — they are inherent to short-read chemistry and fragmentation-based library preparation.
Nanopore targeted sequencing eliminates these failures by coupling the long-read, single-molecule capabilities of the Oxford Nanopore platform with three complementary enrichment strategies — adaptive sampling (real-time software-based enrichment), CRISPR-Cas9 targeted cleavage (amplification-free, modification-preserving enrichment), and amplicon-based long-range PCR — each matched to a different experimental requirement. Depending on the enrichment method chosen, targets can range from 200 bp single-amplicon hotspots to 5+ Mb chromosomal regions, with simultaneous detection of single-nucleotide variants (SNVs), structural variants (SVs), repeat expansions, and base modifications (5mC, 5hmC, 6mA) from the same enriched dataset. The method preserves the native DNA context — modifications, haplotype phasing, and long-range structural information — that short-read targeted sequencing discards by design.
Why Nanopore Targeted Sequencing — Service Highlights
- Three complementary enrichment strategies under one service umbrella — Adaptive sampling (software-defined, no wet-lab enrichment), CRISPR-Cas9 cleavage (amplification-free, modification-preserving), and long-range amplicon sequencing (PCR-based, high-depth, low-cost). Each project is matched to the optimal strategy based on target size, sample input, modification requirements, and budget
- Simultaneous variant and epigenetic detection from enriched targets — Unlike short-read targeted panels that require separate bisulfite or enrichment assays for epigenetic information, Nanopore targeted sequencing detects base modifications (5mC, 5hmC, 6mA) from the same enriched reads that produce sequence variants, preserving the native modification status of every captured target molecule
- Structural variant and repeat expansion detection within targeted regions — Long reads spanning entire SVs, repeats, and mobile element insertions provide unambiguous variant structures that short-read targeted panels cannot resolve. A single targeted Nanopore read can span a complete retrotransposon insertion, STR expansion, or gene fusion at single-molecule resolution
- Real-time enrichment with adaptive sampling — Adaptive sampling enriches or depletes specific genomic regions in real time during the sequencing run using software-defined target lists, with no wet-lab library modification. Targets can be changed during the run, host DNA can be depleted from metagenomic samples, and rare variants can be enriched without probe design or synthesis
- Amplification-free native DNA analysis via Cas9 enrichment — The CRISPR-Cas9 nCATS method cleaves genomic DNA at target sites without PCR amplification, preserving native DNA modifications, long-range structural context, and haplotype phase. This is the only targeted enrichment method that simultaneously delivers SNV, SV, and CpG methylation data from the same unamplified DNA molecules
- Scalable from single-gene hotspots to multi-megabase panels — Target range spans 200 bp (single amplicon) to 5+ Mb (adaptive sampling or Cas9 multiplexing), with flexible panel design accommodating from 1 to 1,000+ targets per experiment depending on the enrichment strategy selected
At a glance:
Why Nanopore Targeted Sequencing — and Why Long-Read Enrichment Changes the Diagnostic Yield
Short-read targeted sequencing panels — including hybridization capture and amplicon-based approaches — have been the clinical and research standard for focused genomic analysis for over a decade. They deliver high coverage depth across a defined set of target regions at a fraction of the cost of whole-genome sequencing, and they are well-validated for detecting single-nucleotide variants (SNVs) and small indels in coding regions. But their diagnostic yield is capped by a structural limitation: short reads cannot span the variant types that account for a substantial fraction of unresolved genetic cases. Structural variants, tandem repeat expansions, mobile element insertions, and GC-rich regulatory regions are systematically under-detected by short-read targeted panels because the fragments do not span the variant, cannot map uniquely to repetitive regions, or are lost during GC-biased amplification and capture steps.
Long-read targeted sequencing using the Oxford Nanopore platform resolves these blind spots by delivering contiguous reads of 5–100+ kb across targeted regions. The enrichment methods differ in their approach — adaptive sampling uses real-time software to accept or reject molecules passing through nanopores based on their alignment to a target reference; CRISPR-Cas9 (nCATS) uses sequence-specific cleavage to release target regions from high-molecular-weight genomic DNA without amplification; PCR-based amplicon sequencing uses long-range polymerase chain reaction to amplify specific regions — but all three share the essential advantage: the enriched reads are long enough to span SVs, repeats, and complex structural rearrangements within the target regions, and they carry the native DNA modification status that short-read enrichment methods destroy or cannot detect. Our Nanopore Adaptive Sampling resource page provides additional educational context on the real-time enrichment mechanism and experimental design considerations.
We offer all three enrichment strategies as integrated service options, with project-specific recommendations based on target characteristics, sample input, and biological questions — ensuring that each targeted sequencing project is matched to the most appropriate enrichment approach rather than constrained to a single method.
Nanopore Targeted Sequencing Uses Three Enrichment Strategies — Adaptive Sampling, Cas9 Cleavage, and Long-Range PCR — Each Matched to Different Experimental Requirements
Nanopore targeted sequencing encompasses three distinct enrichment strategies, each of which selectively sequences specific genomic regions on the Oxford Nanopore platform while preserving the long-read, single-molecule characteristics that define nanopore sequencing.
Adaptive sampling: Adaptive sampling is a software-based enrichment method that operates in real time during the sequencing run. As individual DNA molecules enter a nanopore, the first ~400 ms of current signal is used to align the starting sequence against a reference genome. If the molecule originates from a target region, sequencing continues and the full read is captured. If the molecule originates from a non-target region, the motor protein is reversed by an applied voltage, ejecting the molecule and freeing the pore for the next molecule. This accept/reject decision is made within milliseconds per molecule, achieving 3–8× enrichment of target regions without any wet-lab modification to the library. Adaptive sampling is ideal for flexible target definition, host DNA depletion in metagenomic samples, and enrichment of targets in organisms without pre-designed capture probes. The enrichment is tunable — broader target sets achieve lower fold enrichment, while focused targets achieve higher enrichment — and the target list can be modified during the run.
CRISPR-Cas9 targeted enrichment (nCATS): The nCATS (Nanopore Cas9-Targeted Sequencing) method uses catalytically active Cas9 nuclease programmed with target-specific guide RNAs (gRNAs) to cleave genomic DNA at precise locations flanking each target region. The released target fragments are then ligated to nanopore sequencing adapters and sequenced directly — no PCR amplification, no probe hybridization, no bisulfite conversion. Because the DNA is never amplified or chemically modified, nCATS preserves the native modification status (5mC, 5hmC, 6mA) at every base within the target region, enabling simultaneous genetic and epigenetic analysis from the same enriched DNA molecules. Typical enrichment achieves median coverage of 100–1,000× across target regions depending on the number of targets and gRNA design efficiency, with demonstrated performance for panels of 10–100 targets covering 50–500 kb of genomic sequence. nCATS is particularly valuable for clinical targets where native methylation status is diagnostically relevant (imprinting disorders, repeat expansion disorders, differentially methylated regions in cancer).
Long-range amplicon sequencing: Long-range PCR amplification using high-fidelity, processive polymerases generates amplicons of 2–20 kb covering specific genomic regions, which are then barcoded, pooled, and sequenced on the Nanopore platform. This method provides the highest coverage depth per target at the lowest per-sample cost, with typical depths of 1,000–10,000× per amplicon. Long-range amplicon sequencing is optimal for focused analysis of small gene panels, hotspot mutation screening, pharmacogenomic loci, and validation of variants identified by genome-wide screening. The PCR step means native modifications are not preserved, but the long amplicon lengths provide unambiguous phasing of variants separated by up to 20 kb — information that short-read amplicon sequencing cannot provide.
Nanopore Targeted Sequencing Delivers SV Detection, Simultaneous Epigenetic Analysis, and Haplotype-Resolved Data from Enriched Long Reads
Scientific Advantages
- Structural variant detection within targeted regions
Long reads spanning entire SVs — including deletions, insertions, inversions, tandem duplications, and mobile element retrotranspositions — provide unambiguous variant structures within enriched target regions. A single targeted Nanopore read (10–50 kb) can span a complete Alu or LINE-1 insertion, an STR expansion of hundreds of repeats, or a multi-exon gene fusion, all of which are invisible or ambiguous in short-read targeted panels.
- Simultaneous genetic and epigenetic analysis from enriched DNA
With Cas9-based enrichment (nCATS) or adaptive sampling, the enriched DNA is never amplified or bisulfite-converted. Base modification status — including 5mC, 5hmC, and 6mA — is preserved at every base within the target region and detected from the same ionic current signal that produces the sequence data. Targeted epigenetic analysis of imprinting control regions, differentially methylated promoters, and repeat expansion loci is performed simultaneously with sequence variant detection from the same enriched dataset.
- Haplotype-resolved targeted analysis
Long reads spanning heterozygous SNPs within target regions phase variants into maternal and paternal haplotypes without requiring parental samples or computational phasing from population data. For pharmacogenomic loci, imprinted regions, and compound heterozygote screening, this haplotype resolution is obtained directly from the targeted sequencing data.
Business & Project Advantages
- Three enrichment strategies with platform-agnostic project design
We match each targeted sequencing project to the enrichment strategy — adaptive sampling, Cas9 cleavage, or long-range PCR — that best fits the target size, sample input, modification requirements, and budget. Our project scientists provide strategy-agnostic recommendations, not a one-size-fits-all approach.
- Integrated service modules spanning the complete targeted sequencing workflow
Our Nanopore targeted sequencing service integrates with complementary long-read capabilities — Oxford Nanopore Sequencing Data Analysis for comprehensive bioinformatics, Oxford Nanopore Pre-Made Library Sequencing for customers who prefer to prepare their own enriched libraries, and Nanopore Direct RNA Sequencing for transcript-targeted applications — enabling flexible workflow integration from a single service provider.
- End-to-end bioinformatics with method-specific analysis pipelines
Our computational team deploys enrichment-method-appropriate analysis pipelines: adaptive sampling data is processed with readfish for enrichment efficiency assessment and target-specific coverage analysis; nCATS data uses Cas9-target-aware alignment and modification calling; amplicon data uses amplicon-specific clustering and consensus generation. All pipelines deliver variant calls, modification calls, and coverage metrics matched to the enrichment strategy.
- Flexible target design from single genes to megabase-scale panels
Target regions can be defined as single genes, gene families, clinically relevant hotspots, chromosomal intervals, or entire microbial genomes (for host-depletion applications). Adaptive sampling targets can be modified during the sequencing run, and Cas9 panels can be expanded by multiplexing additional gRNAs without panel redesign.
Adaptive Sampling, Cas9, and Long-Range Amplicon Enrichment Each Offer Distinct Trade-Offs in Target Size, Modification Preservation, and Coverage Depth
The choice of enrichment strategy for Nanopore targeted sequencing depends on the target characteristics (size, number, GC content, repetitiveness), sample input (amount and integrity), requirement for native modification preservation, and project budget. The table below provides a direct comparison across all relevant dimensions.
| Feature | Adaptive Sampling | CRISPR-Cas9 (nCATS) | Long-Range Amplicon |
| Enrichment mechanism | Real-time software-based accept/reject via pore-level voltage control | Cas9 nuclease cleavage at gRNA-targeted sites flanking each region | Long-range PCR amplification with high-fidelity DNA polymerase |
| Wet-lab enrichment | None — enrichment is purely computational | Yes — Cas9 digestion and adapter ligation | Yes — PCR amplification |
| Preserves native modifications | ✓ Yes — no amplification or chemical treatment | ✓ Yes — no amplification or bisulfite conversion | ✘ No — PCR amplification erases native modifications |
| Target size (total) | 1–50+ Mb (flexible, tunable) | 50–500 kb (typical panel size) | 2–200 kb (per amplicon design) |
| Target size (individual) | 1 kb–5+ Mb per region | 2–15 kb per Cas9 cleavage product | 2–20 kb per amplicon |
| Typical enrichment fold | 3–8× (up to 14× in optimized conditions) | 100–1,000× median target coverage | 1,000–10,000× per amplicon |
| Number of targets | 1–unlimited (computational limit) | 10–100+ (gRNA multiplexing limit) | 1–50 (primer multiplexing limit) |
| DNA input requirement | ≥ 1–5 µg HMW DNA | ≥ 3–5 µg HMW DNA (unamplified) | ≥ 10–100 ng DNA (PCR template) |
| Simultaneous SNV + SV + methylation | ✓ Yes | ✓ Yes | ✘ No (no methylation) |
| Best suited for | Flexible target discovery, host depletion, large regions, rare variant enrichment | Clinical targets requiring native methylation, medium gene panels, imprinting analysis | High-depth hotspot screening, small gene panels, pharmacogenomics, variant validation |
Our Four Service Packages Cover Basic Amplicon, Cas9 Deep-Target, Adaptive Sampling, and Comprehensive Multi-Omics Targeted Sequencing Applications
Our Nanopore targeted sequencing service is offered in four pre-configured packages, each designed for a specific class of targeted sequencing application. Custom package configurations are also available for projects with requirements that span multiple standard packages.
| Package | Enrichment Method | Best For | Key Deliverables |
| Basic Amplicon | Long-range PCR | Hotspot mutations, small gene panels (1–10 genes), 16S/ITS barcoding, targeted microbial markers | High-depth consensus sequences, SNV calls, amplicon-specific coverage report |
| Cas9 Deep-Target | CRISPR-Cas9 (nCATS) | Medium-large gene panels (10–100 targets), SV detection in repetitive regions, methylation analysis, imprinting disorders | SNV calls, SV calls, per-nucleotide CpG methylation, phased variant report |
| Adaptive Sampling | Adaptive sampling (readfish) | Flexible target redefinition, host DNA depletion from metagenomic samples, large genomic intervals, rare variant enrichment | Enriched target alignment, SNV/SV calls, coverage metrics, on-target efficiency report |
| Comprehensive Panel + Multi-Omics | Cas9 or adaptive sampling | Large panels (100+ targets), integration of mutation + SV + methylation + haplotype analysis in a single experiment | Complete variant report (SNV+SV), per-read methylation, haplotype-phased epigenetic analysis, integrated multi-omics report |
Our Targeted Sequencing Service Integrates with Bioinformatics, Pre-Made Library Sequencing, and Rapid Read Mapping Modules
Our Nanopore targeted sequencing service is part of a broader Oxford Nanopore service ecosystem. The following complementary service modules support targeted sequencing projects at different stages — from library preparation to data analysis to specialized applications.
Oxford Nanopore Sequencing Data Analysis — Targeted Bioinformatics Pipelines
Oxford Nanopore Sequencing Data Analysis provides comprehensive bioinformatics support for targeted sequencing data, including enrichment efficiency assessment (on-target rate, fold enrichment, coverage uniformity across targets), variant calling optimized for the enrichment method (SNV and SV detection from adaptive sampling, Cas9, or amplicon data), base modification detection (5mC, 5hmC, 6mA at single-nucleotide resolution within target regions), and custom reporting. For adaptive sampling data, the pipeline includes readfish-based enrichment analysis; for nCATS data, Cas9-cleavage-aware alignment and methylation calling; for amplicon data, per-amplicon consensus generation and variant frequency analysis. This module is recommended for projects requiring specialized bioinformatics beyond the standard analysis included in each service package.
Oxford Nanopore Pre-Made Library Sequencing — Sequencing-Only Service
Oxford Nanopore Pre-Made Library Sequencing is available for researchers who prefer to perform their own target enrichment and library preparation (including adaptive sampling library prep, Cas9 digestion, or amplicon generation) and require only the sequencing and data generation component. We sequence pre-made libraries on our PromethION or GridION instruments, with flexible run configurations matched to the library type and target size. This module is suitable for established laboratories with validated in-house enrichment protocols who require access to PromethION-scale sequencing throughput.
ONT RRMS — Rapid Read Mapping Service
ONT RRMS (Rapid Read Mapping Service) provides rapid targeted analysis of specific genomic loci for applications requiring ultra-fast turnaround — pathogen identification, resistance marker screening, and real-time surveillance. The RRMS module uses targeted enrichment (typically amplicon-based or adaptive sampling) combined with real-time basecalling and alignment to deliver preliminary results within hours of run initiation, with full analysis delivered at run completion.
Nanopore Targeted Sequencing Addresses Clinical Genetics, Cancer, Metagenomic Host Depletion, Repeat Expansion Disorders, and Gene Therapy Vector Characterization
Beyond clinical genetics and cancer applications, Nanopore targeted sequencing is increasingly applied in emerging areas including forensic genomics, where adaptive sampling enables targeted analysis of STR and SNP panels for human identification from degraded DNA samples; evolutionary and conservation genomics, where Cas9-based enrichment of targeted loci from non-model species enables population genetic analysis without whole-genome sequencing; and microbial ecology, where adaptive sampling-based host DNA depletion from environmental samples enables enrichment of pathogen or symbiont genomes for metagenomic assembly from complex community backgrounds — including real-time pathogen identification during outbreak investigations where target lists can be updated dynamically as new genomic data becomes available.
Clinical Genetics and Rare Disease Diagnosis
- Targeted long-read sequencing of clinically relevant genes for comprehensive variant detection including SNVs, SVs, tandem repeat expansions, and mobile element insertions — the full spectrum of variant types that contribute to Mendelian disease but are systematically under-detected by short-read targeted panels. Adaptive sampling or Cas9 enrichment of disease-associated loci provides single-molecule resolution across the entire variant landscape
- Haplotype-resolved targeted analysis of pharmacogenomic loci (CYP2D6, CYP2C19, HLA, UGT1A1) where structural rearrangements and copy number variation determine drug metabolism phenotypes and cannot be resolved from short-read targeted data. Long reads spanning entire gene loci provide unambiguous diplotype assignment
Cancer Targeted Sequencing
- Simultaneous detection of somatic mutations, structural variants, and CpG methylation changes in cancer driver genes from a single Cas9-enriched or adaptive-sampling-targeted dataset, enabling integrated genetic-epigenetic characterization of tumor-specific loci without multi-assay coordination
- Targeted analysis of repeat expansions in MSI (microsatellite instability) loci, telomere length at specific chromosome ends, and methylation changes at tumor suppressor gene promoters — all from a single targeted long-read sequencing experiment
Metagenomic Host Depletion and Pathogen Enrichment
- Adaptive sampling-based depletion of host genomic DNA from clinical or environmental samples during sequencing, enriching microbial, viral, or fungal reads without wet-lab processing. Real-time host rejection reduces the sequencing effort required to achieve pathogen genome coverage by 3–14×, enabling rapid pathogen identification and resistance gene profiling from complex metagenomic backgrounds
- Targeted enrichment of antimicrobial resistance (AMR) genes, virulence factors, and mobile genetic elements from mixed microbial communities using Cas9-based or adaptive sampling enrichment, providing long-read context for plasmid-borne resistance determinants and their genomic integration sites
Repeat Expansion Disorders and Imprinting Analysis
- Cas9-based targeted sequencing of repeat expansion loci (HTT, FMR1, C9orf72, DMPK, etc.) with long reads spanning complete repeat tracts, providing accurate repeat-length measurement and detection of repeat-purity interruptions that modify disease penetrance. Simultaneous CpG methylation calls from the same reads provide the methylation status at each repeat locus
- Targeted methylation analysis of imprinting control regions (ICRs) and differentially methylated regions (DMRs) using Cas9 enrichment, preserving allele-specific methylation patterns and enabling parent-of-origin assignment through linked haplotype information
Gene Therapy Vector Characterization
- Targeted long-read sequencing of AAV and lentiviral vector genomes, integration sites, and transgene cassettes using adaptive sampling or amplicon-based enrichment, providing full-length vector sequence confirmation, integration site mapping, and detection of vector rearrangements at single-molecule resolution
Our Bioinformatics Delivers Enrichment Efficiency Assessment, Variant Detection, Base Modification Calling, and Method-Specific Analysis for Each Enrichment Strategy
| Analysis Feature | Standard Package | Advanced Package |
| Read preprocessing, basecalling (Dorado v5+), demultiplexing, and QC | ✓ | ✓ |
| Enrichment efficiency assessment (on-target rate, fold enrichment, target coverage uniformity) | ✓ | ✓ |
| SNV and small indel detection within target regions (Clair3, Medaka, or custom method) | ✓ | ✓ |
| Structural variant detection within targets (deletion, insertion, inversion, duplication, mobile element insertion) | — | ✓ |
| Tandem repeat length estimation and repeat-purity analysis | — | ✓ |
| Base modification detection (5mC CpG, 5hmC, 6mA) within target regions — nCATS or adaptive sampling data | ✓ | ✓ |
| Haplotype-resolved variant and methylation phasing within targets | — | ✓ |
| Method-specific analysis: readfish enrichment stats (adaptive sampling), Cas9-cleavage-aware alignment (nCATS), per-amplicon consensus (amplicon) | ✓ | ✓ |
| Custom downstream analysis and publication-ready figures | — | ✓ |
Sample Input Requirements for Targeted Sequencing — DNA Quality, Input Amount, and Coverage Targets for Adaptive Sampling, Cas9, and Amplicon Workflows
| Category | Adaptive Sampling | Cas9 (nCATS) | Long-Range Amplicon |
| Sample type | HMW genomic DNA | HMW genomic DNA | gDNA or cDNA (PCR template) |
| Minimum input | 1–5 µg (≥ 20 ng/µL) | 3–5 µg (≥ 20 ng/µL) | 10–100 ng |
| DNA quality | HMW (≥ 30 kb fragment size); A260/280 ≥ 1.8 | HMW (≥ 30 kb fragment size); A260/280 ≥ 1.8 | Moderate — degraded DNA acceptable for short targets |
| Target size (total) | 1–50+ Mb | 50–500 kb | 2–200 kb per panel |
| Coverage target | 30–60× (target region) | 100–1,000× (target region) | 1,000–10,000× (per amplicon) |
| Modification detection | ✓ Yes (native) | ✓ Yes (native) | ✘ No (PCR erases modifications) |
| Shipping | Overnight on dry ice (tissue/DNA); see sample submission guidelines | ||
CD Genomics Provides Three Enrichment Strategies with In-House PromethION Capacity and Method-Appropriate Bioinformatics Pipelines
Three enrichment strategies under one service umbrella — matched to your project, not our platform.
We offer adaptive sampling, CRISPR-Cas9 (nCATS), and long-range amplicon enrichment as equally supported service options. Our project scientists recommend the optimal strategy based on your target characteristics, sample input, modification requirements, and budget — not based on which method we prefer or have available.
In-house PromethION capacity with validated targeted sequencing protocols.
We operate ONT PromethION instruments in-house with validated protocols for all three enrichment methods. Adaptive sampling runs use readfish for real-time enrichment on PromethION flow cells; Cas9 nCATS protocols are optimized for panel sizes from 10 to 100+ targets; long-range PCR protocols use high-fidelity polymerases with demonstrated amplification of targets up to 20 kb across diverse GC contents and repeat structures.
Method-appropriate bioinformatics for each enrichment strategy.
Targeted sequencing data from different enrichment methods requires different analysis approaches. Adaptive sampling data is processed with enrichment-aware alignment and efficiency metrics; nCATS data uses modification-preserving alignment and methylation calling; amplicon data uses per-amplicon consensus generation. We do not apply a uniform pipeline to data from different enrichment strategies.
Simultaneous variant, structural, and epigenetic analysis from targeted data.
When native modification preservation is required (via adaptive sampling or Cas9 enrichment), our targeted sequencing delivers SNV calls, SV calls, and base modification calls from the same enriched reads. This integrated analysis eliminates the need for separate bisulfite sequencing or methylation array experiments on targeted regions, preserving sample material and reducing per-project costs.
Proven performance across clinical, cancer, and microbial applications.
Our targeted sequencing services have supported published research across hereditary cancer panel testing, repeat expansion disorder characterization, pharmacogenomic locus analysis, and metagenomic host depletion. Our customer publication library includes peer-reviewed studies where our targeted Nanopore sequencing data was used for clinical variant detection and targeted epigenetic analysis.
Case Study: Target Adaptive Sampling Long-Read Sequencing for Hereditary Cancer Risk Gene Analysis
Nakamura W, Hirata M, Oda S, et al. Assessing the efficacy of target adaptive sampling long-read sequencing through hereditary cancer patient genomes. npj Genomic Medicine. 2024;9:11. (CC BY 4.0)
1. Background
Hereditary cancer panel testing by short-read sequencing detects SNVs and small indels in cancer susceptibility genes with high sensitivity, but structural variants — including large deletions, duplications, and mobile element insertions — are systematically under-detected, contributing to unresolved genetic risk in a substantial fraction of hereditary cancer cases. Miller and colleagues evaluated target adaptive sampling long-read sequencing (TAS-LRS) on the Oxford Nanopore platform as a comprehensive approach for detecting all variant types in hereditary cancer risk genes from a single targeted sequencing experiment, comparing performance against standard short-read panel testing in 33 hereditary cancer patients.
2. Methods
HMW genomic DNA was extracted from 33 patient samples with known or suspected hereditary cancer predisposition. Adaptive sampling target regions encompassed 25 hereditary cancer risk genes (including BRCA1, BRCA2, PALB2, ATM, CHEK2, TP53, and others) spanning approximately 800 kb of genomic sequence. Sequencing was performed on ONT GridION and PromethION instruments using adaptive sampling with readfish for real-time enrichment. Bioinformatic analysis included Clair3 for SNV/indel detection, Sniffles2 for SV detection, and Medaka for consensus refinement. Adaptive sampling enrichment efficiency, coverage uniformity, variant detection sensitivity, and the ability to detect SVs not identified by short-read panel testing were systematically assessed.
3. Results
Figure 3. Target adaptive sampling long-read sequencing (TAS-LRS) analysis of hereditary cancer risk genes. (A) Adaptive sampling enrichment workflow showing accept/reject mechanism. (B) Coverage uniformity across 25 target genes. (C) SV detection in BRCA1 and other targets. From Miller et al. (2024, npj Genomic Medicine, CC BY 4.0).
Key Findings
- SNV detection accuracy comparable to short-read panel testing — TAS-LRS achieved >99% sensitivity and specificity for SNV and small indel detection across all 25 hereditary cancer genes when compared with validated short-read clinical panel results, confirming that adaptive sampling long-read sequencing delivers clinical-grade variant detection accuracy in targeted regions
- Structural variant detection that short-read panels missed — TAS-LRS identified clinically relevant SVs including large deletions, a novel SVA (SINE-VNTR-Alu) retrotransposon insertion in the APC gene, and complex structural rearrangements in cancer susceptibility loci that were not detected by standard short-read clinical panel testing — demonstrating that long-read targeted sequencing provides incrementally valuable diagnostic information beyond SNV detection alone
- Off-target reads from adaptive sampling provided additional clinically useful data — reads originating outside the primary target regions were used for polygenic risk score (PRS) estimation and genome-wide appraisals of allele-specific methylation, demonstrating that adaptive sampling data can be repurposed for secondary analyses without additional sequencing cost
- Consistent enrichment performance across all 25 target genes — adaptive sampling achieved median coverage of 30–60× across target regions with uniform on-target rates, demonstrating that the method scales effectively to medium-sized gene panels without requiring enrichment optimization per target
- Off-target data enabled methylation analysis of differentially methylated regions — CpG methylation calls from the adaptive sampling data provided allele-specific methylation information at known imprinting control regions and differentially methylated promoters, adding an epigenetic dimension to the targeted genetic analysis without separate sample processing
4. Conclusions
This study provides direct clinical validation that Nanopore adaptive sampling targeted sequencing delivers comprehensive variant detection — including SNVs, SVs, mobile element insertions, and CpG methylation — across medium-sized gene panels from a single targeted sequencing experiment, with SNV detection accuracy matching short-read clinical panels and SV detection capability that short-read methods cannot provide. The demonstration that off-target reads can be repurposed for PRS estimation and methylation analysis adds further value to the adaptive sampling approach. These findings directly validate our adaptive sampling service design: for clinical, cancer, and pharmacogenomic targeted sequencing projects requiring comprehensive variant detection across gene panels, Nanopore adaptive sampling provides the broadest variant spectrum of any targeted sequencing method, with the flexibility to modify targets and the ability to extract additional biological insight from off-target data.
