Nanopore sequencing is a cool new DNA-reading technology that works like a microscopic scanner. It pulls DNA strands through tiny protein holes while detecting electrical signals to read the genetic code in real time. The best part? It can read super long DNA sections (over 100,000 letters!) all at once, helping scientists study complex genetic areas that were hard to analyze before. It even detects special chemical tags on DNA that control gene activity. While earlier versions made more mistakes, newer models are over 99% accurate thanks to smart upgrades. Doctors use it for quick virus detection during outbreaks and to study cancer DNA. What's really amazing is its portability - some devices are small enough to use anywhere, from hospitals to rainforests! This technology is changing how we understand DNA in medicine and science.
This article primarily explores how nanopore sequencing enables real-time, long-read variant calling and its transformative applications in genomics, clinical diagnostics, and epigenetics.
What is Nanopore-Based Variant Calling
Revolutionary Sequencing Approach
Nanopore sequencing is an exciting new way to read DNA that works like a tiny scanner. Picture pushing a DNA strand through a tiny hole, called a "nanopore." A machine reads its genetic code by sensing small electrical changes. It's like scanning a barcode at the store! What makes this special is its ability to read long DNA sections at once. This helps scientists solve genetic puzzles that were once too tricky. It's quick enough to provide results right away. Some versions are tiny, so they can be used anywhere, even in the jungle! It reads the DNA letters and finds special chemical tags. These tags work like sticky notes, showing cells which genes to use. Doctors use it to study diseases, track viruses, and even help solve crimes. Today's nanopore sequencers are much more accurate. Early versions had mistakes, but now smart computer programs double-check the results. This amazing technology is making DNA research faster, more portable, and way more fun!
Nanopore Calling's Underlying Technology
Nanopore sequencing detects small electric changes when DNA strands move through tiny holes. Each DNA piece creates a unique signal that the machine reads like a fingerprint. Oxford Nanopore creates devices that read very long DNA strands. These strands can have thousands to millions of letters. The process does not require steps like copying or tagging the DNA.
Transformative Features of Nanopore Calling
Nanopore sequencing is special because it can: 1) read very long pieces of DNA at once, and 2) show results in real time. This helps scientists see tricky parts of DNA that other methods miss, like repeating sections or big changes in the genetic code. Reading these long sections helps spot genetic differences more accurately. It can detect small single-letter changes and major DNA rearrangements.
Figure 1. The comparative analysis of different sequencing platforms and variant calling pipelines.( Shikov, 2021)
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Nanopore Calling's Variant Detection Capabilities
Conquering Complex Genomic Regions
Nanopore sequencing technology has greatly improved our ability to find genomic variants. This applies at many levels of complexity. Nanopore technology offers important advantages for SNPs and small insertions or deletions (indels). It works well in tricky areas like repetitive sequences, pseudogenes, and spots with high GC content. These regions often confuse alignment algorithms that rely on short reads.
Revolutionizing Structural Variant Identification
Nanopore sequencing shines in finding structural variants (SVs). These are genomic changes that cover 50 base pairs or more. They include deletions, duplications, inversions, and translocations. Larger genomic rearrangements are linked to many genetic disorders and cancer. However, short-read technologies struggle to detect them fully. Nanopore's multi-kilobase reads cover structural variants. They capture breakpoints and rearrangements in one continuous read.
Unraveling Disease-Causing Repeat Expansions
Tandem repeat expansions, which underlie numerous neurological disorders such as Huntington's disease, fragile X syndrome, and various ataxias, represent another area where nanopore sequencing excels. These repetitive sequences can extend to thousands of base pairs and have proven notoriously difficult to resolve with short-read technologies. Nanopore sequencing can traverse entire expanded repeat regions, providing accurate assessments of repeat numbers and configurations that directly impact disease manifestation.
Figure 2. Genome completeness analysis by long-read sequencing only.( Negi .2025)
Nanopore Calling's Real-Time Clinical Applications
Using Nanopore Calling for Infectious Disease Surveillance
Nanopore sequencing has revolutionized pathogen monitoring, especially during the SARS-CoV-2 pandemic. The ARTIC network protocol enabled worldwide genomic surveillance with unprecedented speed, helping track variants of concern and transmission patterns. For bacterial outbreaks, nanopore technology provides complete genomic profiles of antimicrobial resistance within hours rather than days, enabling timely infection control in hospitals.
Using Nanopore Calling for Cancer and Genetic Diagnostics
In oncology, nanopore long-read sequencing detects variants missed by conventional approaches. It comprehensively characterizes large genomic rearrangements in critical genes like BRCA1/2, spanning complex regions that confound short-read methods. The technology also excels in leukemia classification through detection of fusion transcripts and in neurogenetics by accurately sizing trinucleotide repeat expansions associated with conditions like Huntington's disease, providing valuable information for genetic counseling.
Figure 3. Illustration of haplotype-aware variant calling using nanopore variant calling sequencing.( Shafin., 2021)
Technical Challenges in Nanopore Calling
Error Rate and Sequence Context Biases
Despite its revolutionary potential, nanopore sequencing faces higher base-calling error rates (5-15%) compared to short-read technologies (<1%). These errors aren't random but show systematic biases in specific sequence contexts. Signal-to-noise limitations affect accuracy, with electrical current measurements influenced by temperature fluctuations, pore variability, and DNA translocation disruptions.
Problematic Genomic Regions
Homopolymer regions—stretches of identical consecutive nucleotides—create particular difficulties as they produce uniform current signals, making it hard to determine exact nucleotide counts. This frequently results in insertion and deletion errors. Similarly, extreme GC content areas yield lower quality data, with GC-rich regions forming secondary structures that affect DNA movement through the pore, while AT-rich regions produce weaker signals.
Computational Challenges in Nanopore Calling
Processing nanopore data requires sophisticated algorithms to translate electrical signals into sequences—a computationally intensive process. Variant calling algorithms specifically optimized for nanopore data are still evolving rapidly, meaning best practices remain somewhat fluid and require regular reassessment as methods improve.
Accuracy Enhancement Strategies
Nanopore sequencing technology has evolved significantly through several key error reduction approaches. Duplex consensus sequencing, which analyzes both DNA strands to create a consensus read, dramatically reduces random errors to below 1% in optimal conditions. This laboratory-based method effectively identifies and corrects systematic errors in specific sequence contexts.
Complementary computational strategies further improve accuracy. Hybrid approaches combine nanopore's long reads with highly accurate short-read data to polish sequences while preserving structural information. Meanwhile, advanced base-calling algorithms using deep learning techniques continuously improve through training on expanded datasets, significantly enhancing performance in challenging sequences like homopolymer regions. These combined strategies have progressively narrowed the accuracy gap between nanopore and traditional sequencing while maintaining long-read advantages.
Integration with Epigenetic Data in Nanopore Calling
Simultaneous Detection of Genetics and Epigenetics
Nanopore sequencing changes how we analyze genomes. It allows us to detect DNA sequences and epigenetic modifications at the same time in one experiment. Nanopore technology stands out from traditional sequencing methods. It directly detects DNA methylation marks, so there's no need for separate tests for genetic and epigenetic data. This includes 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and N6-methyladenine (6mA). It does this by measuring unique changes in electrical signals as these modified bases pass through the nanopore.
This direct detection method takes advantage of unique electrical signatures from each nucleotide modification. This allows for base-resolution methylation mapping. It avoids the harsh chemical treatments needed in traditional bisulfite sequencing. Removing bisulfite conversion steps keeps the genome intact. It also simplifies preparation and reduces technical errors. This helps prevent sequence coverage losses that often happen with traditional epigenomic methods. New basecalling algorithms and machine learning models improve modification detection. Tools like Nanopolish, Megalodon, and DeepSignal achieve over 90% accuracy in methylation calling. They also keep the original DNA context.
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Applications in Development and Disease
- Developmental Biology Insights
This integrated genomic and epigenomic approach has changed developmental biology research. It allows scientists to link structural variations with methylation changes. These changes can be seen in long continuous reads that cover hundreds of kilobases. Phasing genetic variants with nearby methylation states on the same DNA molecule offers new insights. This helps us see how alleles control epigenetic changes and imprinting. These factors are vital for embryonic development. Nanopore sequencing shows that genetic changes in enhancer regions affect the methylation of nearby promoters. This insight uncovers the cis-regulatory mechanisms that play a role in cellular differentiation and tissue specification.
Nanopore technology in longitudinal studies has mapped changing methylation patterns during development. This method captures temporary epigenetic states that traditional snapshot methods often overlook. These studies found new epigenetic switches that control lineage choices and cell flexibility. This has important implications for regenerative medicine.
- Cancer Genomics Applications
Nanopore technology is valuable in cancer research. It can profile complex structural changes and methylation patterns at the same time. This gives a complete view of tumor genomes. Such information is essential for precision oncology. This dual profiling method spots hidden fusion events. It also reveals unique methylation patterns. These patterns help as diagnostic markers and may be targets for therapy in various cancers. This technology is very useful. It helps identify hypermethylated promoter regions linked to tumor suppressor silencing. At the same time, it detects copy number variations and chromosomal instabilities in the same dataset.
Recent clinical uses of nanopore sequencing allow for real-time cancer monitoring. Changes in mutational profiles and methylation patterns can show treatment resistance. This happens before standard tests find signs of disease progression. Liquid biopsy techniques can now analyze circulating tumor DNA using small samples. This allows us to capture the genetic and epigenetic differences in tumors from blood samples.
- Neurological Disease Mechanisms
Technology has greatly improved our understanding of complex neurological conditions. These conditions arise from both genetic and epigenetic factors. Nanopore sequencing in Fragile X syndrome allows for a complete look at FMR1 gene expansions and methylation in one test. This method helps us understand how CGG-repeat length, methylation density, and the severity of clinical symptoms are linked. Similar approaches show that repeat expansions in different neurodegenerative disorders cause local hypermethylation. This, in turn, harms gene expression and affects neuronal function.
Nanopore methylation profiling has found new epigenetic signatures in brain tissues. This includes tissues affected by Alzheimer's disease, autism spectrum disorders, and psychiatric conditions. The ability to study native DNA from small post-mortem samples keeps fragile epigenetic marks intact. These marks can be lost with amplification methods. This approach reveals disease-related methylation patterns at enhancers and non-coding regulatory elements. Such insights were not available with standard analysis techniques.
References:
- Shikov, A.E., Tsay, et al. (2021). The application of Nanopore sequencing for variant calling on the human mitochondrial DNA. Semanticscholar. https://api.semanticscholar.org/CorpusID:237389707
- Negi, S., Stenton, S. L., et,al. (2025). Advancing long-read nanopore genome assembly and accurate variant calling for rare disease detection. American journal of human genetics, 112(2), 428–449. https://doi.org/10.1016/j.ajhg.2025.01.002
- Shafin, K., Pesout, T., et,al. (2021). Haplotype-aware variant calling with PEPPER-Margin-DeepVariant enables high accuracy in nanopore long-reads. Nature methods, 18(11), 1322–1332. https://doi.org/10.1038/s41592-021-01299-w
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