5 Breakthrough Applications of Nanopore Amplicon Sequencing

Nanopore amplicon sequencing drives rapid detection, real-time sequencing, and precise genome analysis, transforming applications in precision medicine. Researchers now achieve real-time sequencing for rapid detection of pathogens, resistance, and genomes, supporting advanced surveillance and diagnosis. The global long-read amplicon sequencing market will reach USD 1.24 billion in 2024 (A Long-Read Amplicon Sequencing Market report), fueled by demand for high-throughput sequencing in clinical research, rapid and accurate detection, and transcriptomes. Nanopore technologies enable real-time data generation, rapid clinical diagnoses, and comprehensive surveillance of resistance genes in pathogens, improving treatment strategies and outcomes.

• According to a study published in Sci Rep , Nanopore amplicon sequencing reduces pathogen detection time from over 72 hours to just 7 hours, enabling quicker treatment decisions.

• Real-time sequencing supports rapid detection and surveillance of resistance, enhancing public health responses in genomics and transcriptomes.

Key Takeaways

• Nanopore amplicon sequencing drastically reduces pathogen detection time from over 72 hours to just 7 hours, enabling quicker treatment decisions.

• Real-time sequencing enhances public health responses by allowing rapid detection and surveillance of resistance genes in pathogens.

• This technology provides long reads that can span entire gene regions, improving the detection of complex structural variants in cancer research.

• Nanopore sequencing allows for accurate phasing of mutations, helping researchers understand the genetic architecture of cancer genomes.

• Researchers can achieve high-resolution HLA typing, overcoming challenges in analyzing the major histocompatibility complex (MHC) region.

• The technology supports direct detection of epigenetic modifications, enabling simultaneous sequencing and methylation profiling on native DNA.

• Nanopore sequencing enables rapid identification of pathogens and antimicrobial resistance genes, streamlining metagenomic analysis.

Applications of Nanopore Amplicon Sequencing in Genomics: From cancer research to epigenetics, Nanopore sequencing provides breakthrough solutions for structural variant detection, phasing, and methylation analysis

Introduction: The Paradigm Shift from Short-Read to Long-Read Targeted Sequencing

Understanding the Limitations of Traditional NGS (Illumina) for Complex Regions

Short-read sequencing platforms, such as Illumina, have driven many advances in genomics research. However, these technologies often struggle with complex genomic regions. The short reads, typically ranging from 50 to 300 base pairs, cannot span long repetitive sequences or resolve large structural variants. Researchers encounter difficulties when assembling genomes, especially in regions with high GC or AT content. Dephasing effects during sequencing can introduce base-calling errors and increase substitution rates. Uneven read coverage further complicates the analysis of polyploid genomes and long repetitive elements. These limitations hinder accurate detection of copy number variations and make it challenging to reconstruct complete genomic structures.

Note: Many research teams find that traditional NGS platforms cannot provide the resolution needed for advanced studies in cancer research, biomarker discovery, or translational genomics.

A comparison of key metrics between short-read and long-read targeted sequencing technologies highlights these differences:

What is Nanopore Amplicon Sequencing?

Nanopore amplicon sequencing represents a major advancement in targeted sequencing. This technology uses nanopore devices to generate ultra-long reads that can span entire gene regions, repetitive elements, and GC-rich sequences. Researchers can now detect large structural variants, phase alleles, and resolve complex haplotypes in a single sequencing run. Nanopore technologies enable direct sequencing of native DNA, which preserves epigenetic modifications and supports methylation analysis without chemical conversion.

By producing long reads, nanopore amplicon sequencing eliminates assembly gaps and provides telomere-to-telomere coverage. This approach supports high-resolution studies in cancer research, microbial genomics, and therapeutic development. The technology’s optimized strategies enhance the analysis of polyploid genomes and enable accurate variant detection, even in challenging regions. Research teams benefit from real-time data generation, rapid turnaround, and the ability to customize sequencing panels for specific scientific questions.

Sequencing with nanopore technologies empowers scientists to address questions that were previously out of reach. The combination of long-read capability, direct methylation detection, and flexible panel design makes this platform a powerful tool for advanced genomic research.

1. Oncology & Clinical Research: Unlocking Complex Structural Variants (SVs)

Detecting Fusion Genes and Breakpoints with Precision

Cancer research demands robust sequencing technologies for the detection of complex genomic rearrangements. Nanopore amplicon sequencing enables researchers to identify fusion genes and genomic breakpoints with high accuracy. This technology provides long reads that span entire regions of interest, allowing for direct characterization of structural variants. Researchers can now achieve rapid detection of rearrangements, which is essential for translational research and therapeutic development.

The most significant structural variants detected in cancer research using nanopore amplicon sequencing include:

• Large deletions

• Inversions

• Translocations affecting CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes

These applications support the identification of novel biomarkers and the validation of therapeutic targets. The ability to resolve breakpoints at the nucleotide level enhances the precision of variant detection and supports comprehensive analysis of cancer genomes.

The following table highlights improvements in fusion gene and breakpoint detection compared to previous sequencing technologies:

Researchers benefit from streamlined workflows and rapid turnaround, enabling efficient analysis of multiple specimens. The flexibility of nanopore amplicon sequencing allows for the inclusion of additional targets without extensive protocol changes.

Phasing Mutations: Distinguishing Cis vs. Trans Variants in Cancer Genomes

Phasing mutations is critical for interpreting the functional impact of co-occurring variants in cancer research. Nanopore amplicon sequencing provides long-range data that supports accurate phasing of mutations, even when variants are separated by tens of kilobases. This capability allows researchers to distinguish between cis and trans configurations, which is essential for understanding the genetic architecture of cancer genomes.

The table below summarizes the advantages of phasing mutations using nanopore amplicon sequencing:

Researchers can interpret complex genetic findings with greater confidence. The integration of long-range PCR and nanopore sequencing supports comprehensive variant detection and phasing, advancing biomarker discovery and translational research. Nanopore amplicon sequencing empowers research teams to address previously inaccessible questions in cancer genomics, driving innovation in therapeutic development.

2. Immunology: High-Resolution HLA Typing and Haplotype Phasing

Overcoming Homology Challenges in the MHC Region

Immunogenetics research often encounters significant obstacles when analyzing the major histocompatibility complex (MHC) region. The MHC contains highly polymorphic HLA genes, which play a central role in immune response studies. Traditional short-read sequencing platforms struggle to resolve these regions due to extensive sequence homology and structural complexity. Researchers require advanced sequencing methods to achieve accurate variant identification and haplotype phasing within the MHC.

Nanopore amplicon sequencing provides a solution by generating long reads that span entire HLA genes. This approach enables direct detection of variants and unambiguous phasing, even in regions with high sequence similarity. Scientists can now analyze the full sequence of polymorphic HLA loci, overcoming the limitations of short-read technologies. The unrestricted read length of nanopore sequencing facilitates comprehensive analysis of the MHC, supporting applications that demand high-resolution data.

Tip: Long-read sequencing technologies, such as those based on Oxford Nanopore platforms, enable haplotypic resolution and accurate characterization of the MHC region. This capability is essential for resolving the high sequence variability and structural complexities that challenge traditional sequencing methods.

Researchers benefit from rapid, portable, and cost-effective high-resolution HLA typing. The technology eliminates cis-trans ambiguity, allowing for precise haplotype phasing and variant detection in immunogenetics studies.

Achieving Full-Length Allele Resolution for Transplant Compatibility

Full-length allele resolution represents a critical advancement in HLA typing. Nanopore amplicon sequencing captures the entire sequence of HLA genes, including distant single nucleotide polymorphisms (SNPs) that are difficult to phase with short reads. This comprehensive approach reduces ambiguity in allele assignment and enhances the accuracy of genotype determination.

High-resolution sequencing directly impacts the assessment of donor-recipient compatibility in transplantation research. By resolving phase ambiguities, scientists can determine specific alleles with greater confidence. This level of detail improves the reliability of matching studies and supports the development of new immunogenetics applications.

Nanopore sequencing also enables direct detection of epigenetic modifications within HLA genes. This feature expands the scope of immunogenetics research, allowing for the integration of methylation analysis with traditional genotyping. Robust error correction algorithms further enhance the accuracy of HLA typing, ensuring reliable results even in highly polymorphic regions.

Researchers now achieve four-field allele-level resolution, which was previously challenging due to high polymorphism rates and phasing difficulties. The ability to phase distant SNPs and sequence entire HLA genes positions nanopore amplicon sequencing as a superior tool for advanced immunogenetics studies. Scientists gain access to comprehensive data that supports both variant detection and haplotype phasing, driving innovation in immunology research.

3. Gene Editing Validation: Comprehensive On-Target and Off-Target Analysis

Identifying Large Deletions and AAV Integration Sites in CRISPR Experiments

Gene editing technologies, such as CRISPR, have transformed the landscape of genome engineering. Researchers require robust sequencing methods to validate editing outcomes and ensure the integrity of engineered samples. Nanopore amplicon sequencing provides a powerful approach for the detection of large deletions and adeno-associated virus (AAV) integration sites that often escape standard PCR-based methods. This technology delivers long reads that span entire target regions, enabling comprehensive analysis of both on-target and off-target events.

The ability to capture large editing outcomes is critical for accurate characterization of CRISPR experiments. Researchers can directly observe structural changes, including deletions and insertions, across extended genomic regions. The following table compares the effectiveness of different sequencing approaches in identifying large deletions and AAV integration sites:

Researchers benefit from the direct detection of complex genomic rearrangements. Nanopore amplicon sequencing enables the identification of AAV integration sites with high resolution, supporting advanced applications in genome engineering and synthetic biology.

Quantifying Editing Efficiency and Unintended Rearrangements

Precise quantification of editing efficiency remains essential for the validation of gene editing experiments. Nanopore amplicon sequencing supports the detection of intended edits and unintended rearrangements in a single sequencing run. Researchers can analyze the full spectrum of editing outcomes, including small indels, large deletions, and complex structural variants.

This sequencing approach allows for the detection of rare events that may impact the interpretation of experimental results. By generating long reads, researchers can phase multiple edits and distinguish between on-target and off-target modifications. The technology provides real-time data, enabling rapid assessment of editing efficiency and the detection of unexpected genomic changes.

Key advantages of this approach include:

• Direct detection of large deletions and rearrangements

• High sensitivity for rare editing outcomes

• Comprehensive analysis of both on-target and off-target events

• Real-time sequencing for rapid validation

Researchers in genome engineering and synthetic biology rely on nanopore amplicon sequencing for its ability to deliver accurate, high-resolution data. This technology advances the field by enabling the detection and quantification of complex editing outcomes, supporting the development of next-generation gene editing strategies.

4. Epigenetics: Direct Methylation Detection on Native DNA

Simultaneous Sequencing and Methylation Profiling (5mC/5hmC)

Epigenetic modifications, such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), play a critical role in gene regulation and genome stability. Nanopore amplicon sequencing enables researchers to perform simultaneous sequencing and methylation profiling on native DNA. This approach allows direct detection of base modifications without the need for chemical conversion or amplification. Scientists can analyze long DNA strands, which preserves the integrity of epigenetic marks and supports high-throughput applications in complex genomic studies.

Researchers benefit from the ability to distinguish between 5mC and 5hmC signals in a single sequencing run. The technology provides accurate methylation calls across multiple candidate regions, even in large cohorts. The following table summarizes the accuracy of methylation profiling at different coverage depths:

This high correlation demonstrates the reliability of methylation detection, even at moderate coverage. Scientists can scale up the number of amplicons without compromising performance, which supports large-scale epigenetic studies.

Key advantages of this approach include:

• Analysis of longer DNA strands without degradation

• High throughput for large sample sets

• Detection of multiple cytosine modifications in native DNA

• No requirement for DNA amplification or harsh chemical treatment

• Low costs and rapid sample preparation

Researchers can also leverage CRISPR/Cas9-targeted enrichment for cost-effective targeting of specific genomic regions. This flexibility enhances the utility of nanopore amplicon sequencing in advanced epigenetic research.

Eliminating Bisulfite Conversion Bias for More Accurate Epigenetic Data

Traditional bisulfite sequencing introduces biases due to harsh chemical treatment and DNA degradation. Nanopore amplicon sequencing eliminates these issues by enabling direct detection of cytosine methylation in native DNA. The platform measures base modifications through changes in ionic current, which allows real-time sequencing and methylation analysis without prior modification.

The table below highlights the reduction of bisulfite conversion bias:

Researchers can analyze multiple candidate regions simultaneously and customize workflows for large-scale studies. The technology supports high scalability, allowing an increased number of amplicons to be sequenced efficiently. This capability ensures robust and reproducible results for epigenetic applications in research environments.

Tip: Direct methylation detection with nanopore amplicon sequencing provides a streamlined workflow, reduces sample handling steps, and increases data accuracy for complex genomic studies.

5. Advanced Microbiology: Full-Length 16S/ITS and Viral Genomics

Achieving Strain-Level Resolution vs. Genus-Level (V3-V4)

Researchers in metagenomics require high-resolution tools for accurate taxonomic classification. Traditional short-read sequencing often limits metagenomic analysis to genus-level identification, especially when targeting the V3-V4 regions of the 16S rRNA gene. Nanopore amplicon sequencing overcomes this barrier by generating full-length 16S and ITS amplicons, which enables species- and strain-level resolution. This advancement transforms metagenomic sequencing, allowing scientists to distinguish closely related microbial taxa in complex communities.

The following table summarizes key findings from recent metagenomic studies using nanopore platforms:

Metagenomic sequencing with Oxford Nanopore technology achieves read lengths ranging from tens to hundreds of kilobases. This capability supports comprehensive metagenomic analysis, capturing entire gene regions and improving the accuracy of species-level assignments. Although current error rates may not always allow for single-nucleotide resolution, the method provides robust data for quantitative species determination in research applications.

Note: Full-length 16S and ITS sequencing enables researchers to perform quantitative metagenomic analysis, which is essential for studies in environmental microbiology, food safety, and agricultural biotechnology.

Rapid Identification of Pathogens and Antimicrobial Resistance (AMR) Genes

Speed and accuracy are critical in metagenomic sequencing for advanced microbiology research. Nanopore amplicon sequencing delivers rapid detection of microbial taxa and resistance determinants, supporting real-time metagenomic workflows. Researchers can identify pathogens within minutes of sequencing initiation. All predefined antimicrobial resistance genes and plasmids become detectable within one hour using raw sequencing data.

Key advantages of this approach include:

• Identification of microbial taxa within 10 minutes of sequencing

• Detection of all targeted AMR genes and plasmids in under one hour

• Significant reduction in time compared to traditional culture-based metagenomic methods

Metagenomic sequencing with nanopore technology streamlines the detection of rare or unexpected organisms in complex samples. This rapid turnaround accelerates metagenomic analysis, enabling timely insights into microbial community structure and functional potential. Researchers can monitor antimicrobial resistance trends and track the emergence of novel resistance mechanisms with high sensitivity.

Metagenomic sequencing also supports the detection of viral genomes, providing complete coverage for strain-level identification and variant discovery. The ability to generate long reads across entire viral genomes enhances the resolution of metagenomic studies, facilitating the investigation of viral diversity and evolution.

Tip: Nanopore amplicon sequencing empowers research teams to achieve comprehensive metagenomic analysis, from rapid detection to high-resolution taxonomic assignment, in a single streamlined workflow.

Comparison of pathogen detection methods: Traditional culture-based methods vs. Nanopore sequencing, showcasing time, accuracy, and real-time capabilities.

Comparative Analysis: Nanopore vs. Illumina for Amplicon Sequencing

Researchers often evaluate sequencing platforms based on their technical performance in targeted applications. Nanopore and Illumina platforms each offer unique strengths for genomic studies. Understanding the differences in read length, turnaround time, and structural variant sensitivity helps research teams select the optimal tool for their experimental goals.

Nanopore sequencing stands out for its ability to generate ultra-long reads. These reads can span entire gene regions, repetitive elements, and complex structural variants. Illumina sequencing, in contrast, produces shorter reads but achieves high accuracy and depth of coverage. Both platforms support high-throughput workflows, but their technical specifications influence the detection of genomic features.

Structural variant sensitivity varies between the two platforms. Nanopore sequencing achieves near-complete genomes at high depth, supporting the detection of large insertions, deletions, and rearrangements. Illumina sequencing provides higher accuracy for single nucleotide variants and small indels due to its deep coverage. Researchers often choose Nanopore sequencing for comprehensive structural variant detection, while Illumina sequencing remains a strong choice for applications requiring high base accuracy.

The following table summarizes key technical specifications for both platforms:

Table: Read Length, Turnaround Time, and Structural Variant Sensitivity

Note: Nanopore sequencing enables direct detection of complex structural variants and supports phasing across long genomic regions. Illumina sequencing excels in applications that require high accuracy for small variants.

Researchers should consider the specific requirements of their sequencing projects. Nanopore sequencing offers advantages for phasing, methylation analysis, and detection of large genomic changes. Illumina sequencing provides robust performance for high-throughput variant detection and quantitative studies. Both platforms contribute valuable insights to genomic research, but their technical differences guide platform selection for targeted sequencing applications.

Frequently Asked Questions (FAQ) About Amplicon Sequencing

What is the principle behind Nanopore sequencing?

Nanopore sequencing operates by passing single DNA or RNA molecules through engineered nanopores. The system detects changes in ionic current as each molecule translocates. Neural-network basecalling algorithms interpret these signals in real time, converting them into nucleotide sequences. This approach enables direct sequencing and modification detection without amplification or chemical conversion.

What does a typical Nanopore sequencing protocol include?

A standard protocol consists of sample intake and quality control, followed by library preparation. Sequencing proceeds in real time, with basecalling and demultiplexing performed concurrently. The workflow concludes with bioinformatics analysis and structured data delivery. Each step supports high-fidelity detection of variants, methylation, and transcript isoforms.

How long are the reads generated by Oxford Nanopore sequencing?

Nanopore platforms routinely produce long reads, often spanning tens of kilobases. Ultra-long workflows can reach megabase-class molecules. This capability allows comprehensive detection of structural variants, phasing, and full-length transcript analysis.

How is sequencing accuracy reported?

Accuracy is summarized using Q-score distributions. The final accuracy depends on sample quality, sequencing chemistry, read depth, and the analysis pipeline. Researchers receive detailed reports that include accuracy metrics and variant detection rates.

What are the main applications of Nanopore sequencing?

Nanopore sequencing supports de novo assemblies, structural variation analysis, methylome profiling, full-length transcript detection, targeted panels, and metagenomics. These applications enable high-resolution studies in cancer research, biomarker discovery, and therapeutic development.

What is the difference between Cas9 targeted sequencing and adaptive sampling?

Cas9 targeted sequencing uses guide RNAs to enrich specific genomic regions before sequencing. Adaptive sampling enriches or depletes reads during sequencing using software-driven selection. Both methods enhance detection of regions of interest, but adaptive sampling allows dynamic adjustment in real time.

Can Nanopore sequencing detect DNA methylation or RNA modifications?

Nanopore signals support research-grade detection of DNA modifications, including methylation. The platform provides modification-associated signal features, enabling direct analysis of epigenetic marks and transcript variants.

Do bioinformatics pipelines support isoform and fusion detection?

Yes, advanced bioinformatics workflows enable full-length transcript isoform discovery and fusion detection. These tools facilitate comprehensive analysis of complex genomic events and support high-confidence variant calling.

What distinguishes Nanopore Amplicon Sequencing from short-read platforms?

Nanopore Amplicon Sequencing generates long reads that span entire gene regions. This approach enables direct phasing, structural variant detection, and methylation analysis. Researchers gain access to comprehensive data for complex genomic studies.

How does Nanopore technology support variant phasing?

The platform produces reads that cover multiple variants within a single molecule. This capability allows researchers to determine cis or trans configurations, which is essential for interpreting compound mutations in cancer research and biomarker discovery.

Can Nanopore sequencing detect epigenetic modifications directly?

Yes. Nanopore sequencing analyzes native DNA and identifies methylation marks, such as 5mC and 5hmC, without chemical conversion. This direct detection preserves sample integrity and supports high-resolution epigenetic studies.

How does the workflow ensure high data quality?

The workflow includes high-fidelity PCR, stringent quality control, and advanced error correction. Each step minimizes errors and maximizes confidence in variant calls, phasing, and methylation analysis.

Is the workflow customizable for specific research needs?

Yes. CD Genomics offers custom primer design and flexible panel selection. Researchers can target specific genes, regions, or microbial taxa, optimizing the workflow for unique project requirements.

What types of samples are compatible with Nanopore Amplicon Sequencing?

The platform supports high-molecular-weight DNA, RNA, and amplicons from diverse sources. Researchers can analyze human, animal, plant, or microbial specimens, provided the input meets purity and integrity standards.

Does the bioinformatics pipeline support complex variant analysis?

The pipeline enables detection of structural variants, phasing, and methylation. Advanced algorithms filter chimeras and suppress errors, ensuring robust analysis for translational research and therapeutic development.Conclusion: choosing the Right Sequencing Technology for Your Research Goals

Conclusion

Selecting the optimal sequencing platform remains a critical decision for research teams aiming to advance precision medicine. Each technology offers unique strengths, and the choice depends on the specific research objectives, sample types, and the complexity of the genomic regions under investigation.

Long-read third-generation sequencing (TGS) technologies, such as Pacific Biosciences and Oxford Nanopore Technologies, provide significant advantages over short-read technologies. These include the ability to analyze difficult-to-sequence regions, perform native sequencing, and conduct real-time analysis, which are crucial for applications in precision medicine.

Researchers should evaluate several factors when determining the best sequencing approach:

• Read Length Requirements: Long-read sequencing enables comprehensive analysis of structural variants, phasing, and methylation across extended genomic regions. This capability supports high-resolution detection of complex rearrangements and full-length gene characterization.

• Turnaround Time: Real-time sequencing platforms, such as Nanopore, deliver rapid results. This speed accelerates experimental workflows and supports time-sensitive projects.

• Data Output and Accuracy: High-fidelity sequencing ensures reliable variant detection and robust methylation profiling. Advanced error correction pipelines further enhance data quality.

• Customization and Flexibility: Targeted amplicon sequencing allows for the design of custom panels, supporting diverse research goals in cancer research, biomarker discovery, and translational genomics.

A recent study utilizing Nanopore sequencing successfully characterized 37 short tandem repeat loci linked to hereditary neurological disorders, showcasing the technology's clinical potential in precision medicine.

The table below summarizes key considerations for platform selection:

Research teams benefit from the technical superiority of Nanopore amplicon sequencing for complex genomic studies. This platform excels in the detection of large structural variants, phasing of distant mutations, and direct methylation analysis. The ability to perform native sequencing and real-time detection positions Nanopore as a preferred solution for advanced research use only (RUO) applications.

CD Genomics supports scientists with an optimized workflow, expert guidance, and comprehensive bioinformatics analysis. By leveraging the strengths of Nanopore amplicon sequencing, research teams can unlock new insights, accelerate discovery, and address the most challenging questions in genomics.

Tip: Evaluate your research goals, sample types, and the complexity of your target regions before selecting a sequencing platform. The right choice will maximize data quality, detection power, and project efficiency.

Nanopore Amplicon Sequencing delivers transformative impact across applications in oncology, immunology, gene editing, epigenetics, and microbiology. Researchers achieve precise detection and phasing of genetic variants, full characterization of novel isoforms, and robust monitoring of microbial communities. The technology supports high-coverage sequencing, rapid detection, and real-time data analysis, which are essential for advanced research. CD Genomics provides expertise and optimized workflows for seamless adoption. The table below outlines recommended sample preparation for successful implementation:

Researchers can contact CD Genomics for tailored sequencing solutions.