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This report is written by MaltSci based on the latest literature and research findings

What are the applications of long-read sequencing? ​

Abstract ​

The advent of long-read sequencing technologies has significantly transformed the field of genomics, providing researchers with enhanced capabilities to explore the complexities of the human genome and other organisms. Unlike traditional short-read sequencing methods, which are limited in read length, long-read sequencing can generate reads extending tens of thousands of base pairs. This ability is critical for accurately resolving complex genomic regions, structural variations, and repetitive sequences that are often difficult to analyze with shorter reads. The implications of long-read sequencing extend to personalized medicine, as it enhances our understanding of the genetic basis of diseases, leading to more effective therapeutic strategies. Recent advancements in long-read sequencing technologies, particularly those developed by Pacific Biosciences and Oxford Nanopore Technologies, have demonstrated their efficacy in various fields, including cancer genomics, rare genetic disorders, and infectious disease diagnostics. This review systematically explores the diverse applications of long-read sequencing, highlighting its strengths in genome assembly, transcriptome analysis, epigenomics, and clinical research. Key applications include the ability to produce comprehensive genome assemblies, characterize full-length transcripts, and assess DNA methylation and chromatin accessibility. In clinical settings, long-read sequencing shows promise in improving diagnostic rates for complex genetic diseases and facilitating the integration of genomic insights into personalized medicine. As these technologies continue to evolve, their applications are expected to expand, ultimately enhancing our understanding of genetic diversity and disease mechanisms.

Outline ​

This report will discuss the following questions.

  • 1 Introduction
  • 2 Overview of Long-Read Sequencing Technologies
    • 2.1 Types of Long-Read Sequencing Technologies
    • 2.2 Comparison with Short-Read Sequencing
  • 3 Applications in Genome Assembly
    • 3.1 De Novo Genome Assembly
    • 3.2 Structural Variation Detection
  • 4 Applications in Transcriptome Analysis
    • 4.1 Full-Length Transcript Sequencing
    • 4.2 Alternative Splicing Analysis
  • 5 Applications in Epigenomics
    • 5.1 DNA Methylation Studies
    • 5.2 Chromatin Accessibility
  • 6 Applications in Clinical Research
    • 6.1 Disease Association Studies
    • 6.2 Personalized Medicine
  • 7 Conclusion

1 Introduction ​

The advent of long-read sequencing technologies has ushered in a new era in genomics, significantly enhancing our ability to explore the complexities of the human genome and other organisms. Unlike traditional short-read sequencing methods, which typically generate reads of only a few hundred base pairs, long-read sequencing allows for the production of reads that can extend tens of thousands of base pairs. This capability is crucial for accurately resolving complex genomic regions, structural variations, and repetitive sequences that are often challenging to analyze with shorter reads. As such, long-read sequencing has become a powerful tool in various fields of biological and medical research, facilitating a deeper understanding of genome architecture, gene expression, and the molecular underpinnings of diseases.

The significance of long-read sequencing extends beyond mere technical advancements; it holds the potential to transform personalized medicine and enhance diagnostic capabilities. By providing comprehensive insights into genomic structures and variations, long-read sequencing can improve our understanding of the genetic basis of diseases, paving the way for more effective therapeutic strategies and targeted treatments. Recent studies have demonstrated the efficacy of long-read sequencing in elucidating complex genetic diseases, including various forms of cancer, where traditional sequencing methods often fall short in identifying critical alterations [1][2]. Furthermore, as the cost of sequencing continues to decline and throughput increases, the integration of long-read technologies into clinical settings is becoming increasingly feasible [3].

Current research highlights the rapid evolution of long-read sequencing technologies, particularly those developed by Pacific Biosciences and Oxford Nanopore Technologies. These platforms have demonstrated their ability to produce high-quality genomic data, enabling researchers to conduct comprehensive analyses that were previously unattainable [4][5]. The ability to detect structural variants, characterize full-length transcripts, and explore epigenomic landscapes has positioned long-read sequencing as a critical component of modern genomic research [6][7].

This review will systematically explore the diverse applications of long-read sequencing across various domains, structured as follows:

  1. An overview of long-read sequencing technologies, including a comparison with short-read sequencing methods, will provide foundational knowledge on the strengths and limitations of each approach.
  2. The applications of long-read sequencing in genome assembly will be examined, focusing on de novo assembly and structural variation detection, which are pivotal for understanding genomic architecture.
  3. The role of long-read sequencing in transcriptome analysis will be discussed, emphasizing full-length transcript sequencing and alternative splicing analysis, both of which are crucial for comprehending gene expression dynamics.
  4. We will explore the applications of long-read sequencing in epigenomics, highlighting its utility in DNA methylation studies and chromatin accessibility, which are essential for understanding gene regulation.
  5. Finally, the review will address the applications of long-read sequencing in clinical research, including disease association studies and personalized medicine, underscoring its potential to revolutionize diagnostic practices.

In conclusion, as long-read sequencing technologies continue to advance, their applications in biomedical research are expected to expand, ultimately enhancing our understanding of genetic diversity and disease mechanisms. This review aims to provide a comprehensive overview of these technologies, informing researchers and clinicians about their potential to advance both basic and applied sciences in genomics and medicine.

2 Overview of Long-Read Sequencing Technologies ​

2.1 Types of Long-Read Sequencing Technologies ​

Long-read sequencing (LRS) technologies have emerged as transformative tools in genomics, enabling the comprehensive analysis of complex genomic regions that are often inaccessible to traditional short-read sequencing methods. The applications of LRS span various fields, including clinical diagnostics, cancer genomics, rare genetic disorders, and microbial studies, among others.

In clinical settings, long-read sequencing is particularly advantageous for molecular diagnosis and therapy selection. It excels in identifying structural variants (SVs), such as insertions, deletions, and duplications, which are critical for understanding the genetic basis of diseases. For instance, studies have shown that LRS can accurately detect SVs throughout the genome, including regions that are challenging to analyze with short reads, such as repetitive sequences and segmental duplications. This capability is crucial for diagnosing rare genetic disorders, where structural variants are often the underlying cause [8].

In cancer genomics, LRS is rapidly being recognized for its ability to provide a comprehensive view of the genome, transcriptome, and epigenome. It allows for the detection of alterations that previous technologies have overlooked, including complex rearrangements and variations within repetitive regions. The ability to resolve these alterations can lead to improved understanding of cancer biology and potential therapeutic targets [1]. Moreover, LRS facilitates the assembly of complete, gapless genomes, enabling the study of previously intractable regions like centromeres [5].

Additionally, LRS has shown promise in the field of infectious disease diagnostics. It enables the rapid and accurate identification of pathogens directly from clinical samples, providing essential information such as antimicrobial resistance profiles. This capability is particularly valuable in managing infectious diseases where timely and precise identification of the causative agent is critical [9].

In the realm of transcriptomics, long-read RNA sequencing (RNA-seq) has revolutionized the study of transcriptome complexity. By enabling the sequencing of full-length RNA molecules, LRS provides a more accurate characterization of transcript isoforms, allowing researchers to explore variations that cannot be reliably assessed with standard short-read methods [10]. This technology is crucial for understanding gene expression patterns in various diseases, including cancer [11].

The advancements in LRS technologies, particularly those developed by companies like Oxford Nanopore Technologies and Pacific Biosciences, have significantly improved the accuracy, throughput, and cost-effectiveness of sequencing. These developments have opened new avenues for research and clinical applications, with ongoing improvements aimed at addressing challenges such as computational complexity and the need for efficient interpretation of genomic data [3].

In summary, long-read sequencing technologies are versatile tools with a wide range of applications in clinical diagnostics, cancer research, rare disease identification, and microbial genomics. Their ability to provide high-resolution insights into complex genomic regions is poised to enhance our understanding of genetic diversity and disease mechanisms, paving the way for advancements in precision medicine and targeted therapies.

2.2 Comparison with Short-Read Sequencing ​

Long-read sequencing technologies have emerged as a transformative approach in genomics, providing significant advancements in the analysis of complex genomic regions that are often inaccessible with traditional short-read sequencing methods. These technologies, notably from Pacific Biosciences and Oxford Nanopore Technologies, allow for the generation of reads that can span tens to thousands of kilobases, enabling a comprehensive understanding of genomic architecture.

One of the primary applications of long-read sequencing is in the detection and characterization of structural variants (SVs). Long-read technologies have demonstrated a remarkable ability to identify complex structural rearrangements, including large deletions, duplications, and gene fusions, which are critical in understanding various cancers and genetic disorders. For instance, studies have shown that long-read sequencing can successfully uncover SVs in cancer genomes that are often missed by short-read approaches, thus providing insights into the molecular etiology of tumors (Sakamoto et al. 2021; Iyer et al. 2024) [2][7].

Moreover, long-read sequencing enhances the resolution of genomic analyses by facilitating the assembly of diploid genomes, thereby revealing the full spectrum of human genetic variation. This capability is particularly important in population-scale studies and rare disease research, where understanding genotype-phenotype correlations is essential (Rausch et al. 2025) [6]. Additionally, long-read sequencing technologies enable the analysis of epigenetic modifications, such as DNA methylation, directly from the sequencing data, further enriching the understanding of gene regulation and expression (Mahmoud et al. 2025) [12].

In clinical settings, long-read sequencing has been recognized for its potential to improve diagnostic rates for complex genetic diseases. For example, it has been shown to cover regions of the genome that are typically difficult to interpret with short-read sequencing, thus facilitating the identification of previously unrecognized variants associated with diseases (Kobayashi et al. 2022) [13]. Furthermore, the ability of long-read sequencing to detect small variants alongside larger structural changes enhances its utility in precision medicine (Xu et al. 2025) [14].

In comparison to short-read sequencing, long-read technologies offer distinct advantages. Short-read sequencing, while cost-effective and high-throughput, often struggles with repetitive regions and complex genomic structures due to its limited read length (Warburton et al. 2023) [5]. Long reads can bridge these gaps, providing more contiguous assemblies and a clearer picture of genomic architecture. However, long-read sequencing typically has higher error rates and may be more expensive, which poses challenges in achieving sufficient coverage for certain applications (Mahmoud et al. 2025) [12].

In summary, long-read sequencing technologies represent a significant advancement in genomic research and clinical applications, offering enhanced capabilities for detecting structural variants, understanding genetic diversity, and improving diagnostic rates for complex diseases. Their ability to generate comprehensive genomic data positions them as a critical tool in both research and clinical settings, paving the way for deeper insights into human health and disease.

3 Applications in Genome Assembly ​

3.1 De Novo Genome Assembly ​

Long-read sequencing technologies have significantly advanced the field of de novo genome assembly, enabling the reconstruction of complex genomes with improved accuracy and continuity. These technologies, such as those developed by Pacific Biosciences and Oxford Nanopore, provide extended DNA sequences capable of spanning intricate and repetitive regions of genomes, which are often challenging to assemble using traditional short-read sequencing methods.

One of the primary applications of long-read sequencing in de novo genome assembly is its ability to generate more contiguous and complete genome assemblies. For instance, the hybrid assembly approach, which combines long reads for scaffolding with short reads for error correction, has demonstrated remarkable improvements in assembly quality. This method allows for the correction of errors in long read sequences using complementary short reads, resulting in higher-quality assemblies with increased contiguity [15]. Furthermore, tools like Fast-SG utilize ultrafast alignment-free algorithms to construct scaffolding graphs from either short or long reads, facilitating the assembly of large genomes with lower computational demands and costs [16].

Long-read sequencing has proven particularly beneficial for assembling the genomes of non-model organisms, which often exhibit high polymorphism and complex genomic architectures. A study evaluating ten long-read assemblers on Pacific Biosciences datasets revealed that long-read technologies could produce superior quality assemblies compared to those generated using second-generation sequencing [17]. This capability is critical for advancing our understanding of the genetic diversity and evolutionary biology of various species.

Moreover, the application of long-read sequencing extends to the resolution of structural variants (SVs) within genomes, which are essential for linking genotypes to phenotypes in both population-scale studies and rare disease research. The ability to accurately detect SVs has been highlighted as a transformative aspect of long-read sequencing, enabling researchers to explore genomic features that were previously inaccessible [6].

In addition to its role in genome assembly, long-read sequencing has been instrumental in enhancing the characterization of genomic variation at the single-cell level. For example, a study demonstrated that long-read sequencing could provide insights into the genomic architecture of individual cells, revealing a substantial number of high-confidence SVs that were undetectable using short-read approaches [18].

The ongoing advancements in long-read sequencing technologies continue to shape the landscape of genomic research, offering robust solutions for de novo genome assembly and facilitating deeper insights into complex genomic structures and variations. As the technology evolves, it holds promise for addressing the remaining challenges in genome assembly and expanding its applications across diverse biological contexts.

3.2 Structural Variation Detection ​

Long-read sequencing technologies have significantly advanced the field of genomics, particularly in the detection and characterization of structural variations (SVs) within genomes. These applications are crucial for understanding genetic diversity, disease mechanisms, and the complexities of genomic architecture. The following outlines key applications of long-read sequencing in structural variation detection:

  1. Detection of Structural Variants: Long-read sequencing enables the precise identification of various structural variants, including insertions, deletions, duplications, inversions, and complex rearrangements that are often missed by short-read sequencing technologies. This capability is particularly beneficial in cancer genomics, where SVs can play a critical role in tumorigenesis. For instance, studies have demonstrated that long-read sequencing can accurately characterize complex structural aberrations in cancer genomes, such as large deletions and gene fusions, which are essential for understanding the molecular etiology of cancers [19].

  2. Characterization of Non-Coding Regions: Long-read sequencing has proven to be effective in identifying SVs impacting non-coding elements of the genome, which are crucial for regulatory functions. A study involving families with autism spectrum disorders highlighted how long-read sequencing could detect a higher number of SVs in non-coding regions compared to short-read methods, thereby uncovering potential risk factors associated with these conditions [20].

  3. Comprehensive Genome Assembly: The ability of long-read sequencing to produce longer reads allows for better assembly of complex genomic regions that are typically challenging to resolve with short reads. This technology has facilitated the generation of complete, gapless assemblies of human genomes, including repetitive regions that are often problematic for conventional sequencing approaches [5]. Long-read sequencing has also enabled the assembly of diploid genomes, which is critical for revealing the full spectrum of human genetic variation [4].

  4. Population-Scale Studies: Recent advancements in long-read sequencing technologies have allowed for their application in population-scale genomic studies. This has been pivotal in detecting structural variants across large cohorts, which is essential for linking genotypes to phenotypes in various genetic conditions and for understanding the evolutionary dynamics of populations [21].

  5. Improved Diagnostic Rates: Long-read sequencing has shown significant promise in clinical settings, particularly for rare genetic disorders. Its ability to detect structural variants that traditional methods overlook can lead to improved diagnostic rates. For example, in pediatric endocrine disorders, long-read sequencing has identified pathogenic variants that were previously undetected, thereby enhancing the diagnostic yield [22].

  6. Integration with Bioinformatics Tools: The development of specialized bioinformatics tools for long-read data analysis has further enhanced the ability to detect and interpret structural variants. Tools like Sniffles2 and Dysgu have been designed to handle the unique challenges posed by long-read sequencing data, improving the sensitivity and precision of SV detection [8][23].

In summary, long-read sequencing has revolutionized the detection of structural variations by providing comprehensive insights into complex genomic architectures, thereby playing a crucial role in advancing both research and clinical applications in genomics. Its ability to reveal previously inaccessible genomic information positions it as a transformative tool in understanding genetic diversity and disease mechanisms.

4 Applications in Transcriptome Analysis ​

4.1 Full-Length Transcript Sequencing ​

Long-read RNA sequencing (RNA-seq) has emerged as a transformative technology in the field of transcriptome analysis, enabling the comprehensive study of full-length transcripts and providing significant advantages over traditional short-read sequencing methods. The applications of long-read sequencing in transcriptome analysis are vast and impactful, particularly in the context of understanding complex transcript structures, alternative splicing events, and gene expression regulation.

One of the primary applications of long-read RNA-seq is its ability to generate full-length transcript sequences. This capability allows researchers to identify and characterize novel isoforms, alternative splicing events, and fusion transcripts that are often missed by short-read approaches. For instance, long-read sequencing has been utilized to uncover extensive transcript diversity in various biological contexts, such as in human-derived cortical neurons, where a study identified 15,072 transcripts in stem cell-derived cortical neurons alone, alongside numerous differential expression events and usage analyses related to neurodevelopmental and neurodegenerative diseases [24].

Additionally, long-read RNA-seq plays a crucial role in enhancing the accuracy of transcriptome annotation. By enabling the sequencing of longer reads, researchers can more effectively assemble full-length transcripts, thereby improving the completeness and accuracy of transcriptome assemblies. A study on human pancreatic cancer cell lines reported the generation of a high-coverage long-read transcriptome dataset, which not only facilitated isoform discovery but also supported downstream applications such as transcriptome annotation and integration with other omics data [11].

Long-read sequencing technologies also provide insights into transcriptome complexity by enabling the detection of structural variations and epigenetic marks. This is particularly relevant in plant pathology, where long-read sequencing has revolutionized the study of plant and pathogen genomes and transcriptomes, contributing to the understanding of plant-pathogen interactions and disease management [25].

Moreover, the integration of long-read sequencing with novel computational tools enhances its applications in transcriptome analysis. For example, the introduction of TranSigner, a tool designed for accurate read assignment and abundance estimation, demonstrates how long-read data can be effectively utilized to improve transcript quantification [26]. Similarly, the SQANTI tool allows for extensive characterization of long-read transcript sequences, ensuring quality control in full-length transcriptome identification and quantification [27].

In summary, long-read RNA sequencing has a multitude of applications in transcriptome analysis, including the characterization of full-length transcripts, enhancement of transcriptome annotation, investigation of transcript diversity, and integration with advanced computational tools for improved data analysis. These applications are paving the way for a deeper understanding of gene expression regulation and the complexities of transcriptomic landscapes in various biological contexts.

4.2 Alternative Splicing Analysis ​

Long-read sequencing (LRS) technologies have emerged as a transformative tool in transcriptome analysis, particularly in the study of alternative splicing (AS). The inherent ability of LRS to produce full-length transcripts enables researchers to gain deeper insights into the complexity of splicing mechanisms and their implications in various biological contexts.

One significant application of LRS in alternative splicing analysis is its capacity to uncover previously unannotated isoforms and to characterize isoform expression across different cell states. For instance, a study utilizing Oxford Nanopore Technologies for long-read sequencing on the neuroblastoma cell line SH-SY5Y identified a novel transcript of the voltage-gated calcium channel subunit gene, CACNA2D2, revealing differential expression and usage of transcripts during differentiation. This highlights the potential of LRS to facilitate the identification of candidates for future research into state change regulation (Wright et al., 2022) [28].

Furthermore, LRS has been shown to provide a comprehensive mapping of transcript diversity, enabling the identification of extensive transcript variants across different cell types. For example, a study profiling human fibroblasts, induced pluripotent stem cells, and stem cell-derived cortical neurons identified 15,072 transcripts in cortical neurons, with a total of 35,519 differential transcript expression events. This underscores the complexity of transcriptomic regulation and the utility of LRS in advancing our understanding of neurodevelopmental and neurodegenerative diseases (Xu et al., 2025) [24].

LRS also allows for detailed profiling of alternative splicing events at the isoform level, which is critical for understanding the functional consequences of splicing variations. The integration of LRS with computational tools, such as IsoTools, facilitates the reconstruction and quantification of transcripts, enabling researchers to identify alternative splicing events with high accuracy (Lienhard et al., 2023) [29].

Moreover, the use of LRS in combination with other sequencing technologies enhances the resolution of AS analysis. For instance, a comparative study on the use of short-read and long-read sequencing for detecting differential exon usage demonstrated that long-reads excelled in identifying AS events at untranslated regions (UTRs), while short-reads provided better quantification of expression levels. This complementary approach allows for a more comprehensive understanding of AS dynamics during processes such as cell differentiation (Leshkowitz et al., 2022) [30].

In summary, long-read sequencing plays a crucial role in advancing the field of alternative splicing analysis by enabling the identification of novel isoforms, providing insights into transcriptomic complexity, and facilitating the exploration of splicing dynamics in various biological contexts. The integration of LRS with existing technologies and computational frameworks continues to enhance our understanding of the functional implications of alternative splicing in health and disease.

5 Applications in Epigenomics ​

5.1 DNA Methylation Studies ​

Long-read sequencing technologies have significantly advanced the field of epigenomics, particularly in the study of DNA methylation. This approach allows for a more comprehensive understanding of the methylome, which is crucial for elucidating the regulatory mechanisms underlying gene expression and various biological processes. The applications of long-read sequencing in DNA methylation studies can be categorized as follows:

  1. Enhanced Methylation Profiling: Long-read sequencing facilitates the direct detection of DNA methylation patterns at single-base resolution without the need for bisulfite treatment, which can introduce biases and degrade DNA quality. This capability allows researchers to analyze methylation in complex genomic regions, including repetitive elements and transposable elements, which are often challenging to characterize using traditional short-read sequencing methods. For instance, nanopore sequencing can provide insights into the methylation status of 5-methylcytosine and 5-hydroxymethylcytosine across long DNA fragments, enhancing the accuracy of methylation level estimations in transposable elements associated with cancer [31].

  2. Single-Molecule Analysis: Long-read sequencing enables the analysis of DNA methylation at the single-molecule level, revealing heterogeneity in methylation patterns that bulk-level analyses might overlook. Studies have shown that single-molecule methylation profiling can uncover large-scale variations in methylation across the genome, particularly within heterochromatin regions [32]. This granular approach is essential for understanding the epigenetic landscape of various cell types and how they may differ in aging or disease states [33].

  3. Epigenetic Aging Studies: The ability to profile DNA methylation with long-read sequencing has opened new avenues for research into epigenetic aging. By capturing full CpG contexts and accommodating diverse genomic regions, long-read sequencing provides insights into age-associated methylation patterns, facilitating the development of biomarkers for aging [34]. This methodology allows for tracking cellular aging dynamics at a resolution that was previously unattainable.

  4. Clinical Applications: Long-read sequencing has significant potential in clinical settings, particularly in cancer diagnostics. By analyzing DNA methylation changes in cancerous tissues, researchers can identify specific methylation signatures associated with tumorigenesis. For example, a targeted long-read methylation analysis method has been developed that integrates hybridization capture to enhance the detection of methylation patterns in clinical specimens, allowing for haplotype-aware analysis of cancers [35].

  5. Computational Method Development: The growth of long-read sequencing technologies has prompted the development of advanced computational tools to analyze the resulting methylation data. These tools aim to address the unique challenges posed by long-read data, such as alignment and methylation signal calling, enhancing the overall efficiency and accuracy of methylation studies [6].

  6. Integration with Genomic Insights: Long-read sequencing allows for simultaneous measurement of genetic and epigenetic information, providing a holistic view of the genome's regulatory mechanisms. This integration is crucial for understanding complex disease mechanisms, as it helps to link variations in DNA methylation with genetic alterations [36].

In summary, long-read sequencing technologies have revolutionized DNA methylation studies by providing deeper insights into the epigenome, enhancing the resolution of methylation profiling, and enabling new clinical applications. These advancements underscore the transformative potential of long-read sequencing in both basic and applied epigenomic research.

5.2 Chromatin Accessibility ​

Long-read sequencing (LRS) technologies, particularly those developed by Oxford Nanopore Technologies and Pacific Biosciences, have significantly advanced the field of epigenomics, particularly in the study of chromatin accessibility. These technologies provide several key advantages over traditional short-read sequencing methods, enabling a more comprehensive understanding of chromatin dynamics and regulatory mechanisms.

One of the primary applications of long-read sequencing in epigenomics is the simultaneous profiling of chromatin accessibility and DNA methylation. For instance, a study utilizing nanopore sequencing demonstrated the ability to evaluate CpG methylation and chromatin accessibility concurrently on long strands of DNA. This approach, termed nanoNOMe, was applied to four human cell lines, revealing insights into nucleosome occupancy and the combinatorial epigenetic signatures of promoters at single-molecule resolution. Such capabilities allow researchers to generate fully phased human epigenomes, which include chromosome-level allele-specific profiles of chromatin accessibility and methylation, crucial for understanding the differences between cancerous and noncancerous cells [37].

Moreover, long-read sequencing enables high-resolution profiling of chromatin states, as seen in studies employing techniques like Simultaneous Accessibility and DNA Methylation Sequencing (SAM-seq). This method leverages long-read nanopore sequencing to obtain detailed accessibility and methylation landscapes in plant genomes, revealing interactions between chromatin accessibility and DNA methylation across various genomic regions, including genes and transposable elements [38].

In mammalian systems, long-read sequencing has been pivotal in elucidating chromatin accessibility dynamics during early embryonic development. The development of scNanoATAC-seq2 allows researchers to assess chromatin accessibility at single-cell resolution in mouse preimplantation embryos. This technique has uncovered distinct chromatin signatures associated with lineage segregation and provided insights into the reprogramming of chromatin accessibility during early development [39].

Furthermore, long-read sequencing facilitates the exploration of chromatin accessibility in complex systems, such as whole organisms. For example, research in Caenorhabditis elegans has shown that chromatin accessibility changes throughout development can reveal key regulatory elements, emphasizing the importance of studying chromatin dynamics in vivo [40].

The ability of long-read sequencing to directly detect DNA modifications, such as methylation, enhances its utility in profiling chromatin accessibility. This feature allows for the interpretation of molecular phenotypes and provides insights into the parent-of-origin effects of mutations, which is critical for understanding the genetic basis of diseases [6].

In summary, long-read sequencing has revolutionized the field of epigenomics by enabling comprehensive profiling of chromatin accessibility and associated modifications across various biological contexts. Its applications span from basic research in developmental biology to potential clinical applications in cancer genomics, highlighting its transformative potential in understanding the regulatory landscape of the genome.

6 Applications in Clinical Research ​

6.1 Disease Association Studies ​

Long-read sequencing (LRS) has emerged as a transformative technology in clinical research, particularly in the context of disease association studies. This technology addresses significant limitations associated with short-read sequencing, enabling a more comprehensive analysis of complex genomic regions that are often associated with various diseases.

One of the primary applications of long-read sequencing is in the diagnosis of rare genetic disorders. Long-read sequencing effectively detects and clarifies additional disease-associated variants that may be missed by standard diagnostic workflows, particularly for structural variants (SVs), which include insertions, deletions, duplications, inversions, and complex rearrangements. These SVs are crucial in understanding the etiology of many rare diseases, as they often underlie the genetic basis of these conditions. Studies have shown that LRS can yield an additional diagnostic rate of 7%-17% following negative short-read genome sequencing, significantly enhancing the diagnostic yield for undiagnosed rare diseases (Del Gobbo & Boycott, 2025) [41].

Moreover, long-read sequencing is particularly adept at resolving complex genomic regions that are challenging for short-read technologies, such as repetitive sequences and areas with high sequence similarity. This capability allows for a more accurate characterization of genomic alterations, thereby facilitating the identification of disease mechanisms. For instance, long-read sequencing has been successfully applied to detect structural variants in cancer genomes, where it has enabled precise identification of alterations that were previously overlooked by traditional sequencing methods (Sakamoto et al., 2021) [2].

In the context of cancer research, long-read sequencing provides a comprehensive view of genomic, transcriptomic, and epigenomic alterations. This technology not only enhances the detection of SVs but also allows for the evaluation of epigenetic modifications, which are crucial for understanding cancer biology. By utilizing long-read sequencing, researchers can uncover alterations in complex rearrangements and repetitive regions that contribute to tumorigenesis (Li et al., 2025) [1].

Long-read sequencing also plays a pivotal role in elucidating the genetic basis of common diseases by enabling the identification of genetic variants associated with complex traits. The ability to generate high-resolution insights into the human genome has significant implications for linking genotype to phenotype in population-scale studies. Recent advancements in sequencing throughput and computational methodologies are paving the way for integrating long-read sequencing into clinical cohort studies and disease diagnostics (Rausch et al., 2025) [6].

Furthermore, long-read sequencing has applications beyond genetic disorders and cancer. It has been shown to improve the accuracy of pathogen identification in infectious diseases, allowing for the direct sequencing of pathogens from clinical samples and providing critical information such as antimicrobial resistance profiles (Hoang et al., 2021) [9]. This capability is particularly important for managing infectious diseases, where rapid and accurate identification of causative agents is essential for effective treatment.

In summary, long-read sequencing is revolutionizing clinical research by enhancing the diagnostic yield for rare genetic disorders, improving cancer genomics, and facilitating the identification of disease-associated variants across various conditions. Its ability to provide comprehensive genomic insights positions it as a cornerstone technology for future disease association studies and precision medicine initiatives.

6.2 Personalized Medicine ​

Long-read sequencing (LRS) has emerged as a transformative technology in clinical research, particularly in the realm of personalized medicine. Its unique capabilities address several limitations associated with traditional short-read sequencing, making it a powerful tool for enhancing molecular diagnostics and therapy selection.

One of the primary applications of long-read sequencing in personalized medicine is its ability to provide comprehensive insights into complex genomic regions that are often challenging to analyze with short reads. Long-read technologies, such as those developed by Pacific Biosciences and Oxford Nanopore Technologies, enable the detection of structural variations, long-range haplotype phasing, and the identification of base modifications. This capability is particularly beneficial in cases where short-read sequencing fails to yield conclusive results, as it allows for better detection of structural variants and more accurate resolution of repetitive or non-unique genomic regions (Conlin et al., 2022; Hoang et al., 2021) [9][42].

Furthermore, long-read sequencing has shown significant promise in the molecular diagnostics of constitutional genetic disorders. It has been reported that LRS can increase the diagnostic yield for patients with genetic disorders, thereby facilitating more precise treatment plans and enabling families to make informed decisions regarding care. The technology's ability to uncover previously undetected variants and improve the understanding of genetic disease mechanisms is pivotal for the advancement of personalized medicine (Mastrorosa et al., 2023) [43].

In the context of cancer genomics, long-read sequencing provides a comprehensive view of the genomic landscape, enabling the identification of alterations that previous technologies have overlooked. This includes complex rearrangements and structural variants that are critical for understanding tumor biology and for the development of targeted therapies (Li et al., 2025; Sakamoto et al., 2021) [1][2]. The ability to analyze the epigenomic status surrounding structural variants further enhances the understanding of their functional implications in cancer progression.

Long-read sequencing is also poised to play a significant role in the diagnosis and management of rare diseases. Its application can help resolve cases that remain unsolved after conventional testing methods, thus increasing the diagnostic yield and expediting the identification of genetic causes (Xu et al., 2025) [14]. Additionally, LRS can provide valuable insights into the genetic basis of complex diseases by allowing for the characterization of large structural variations and repeat expansions that are often implicated in disease etiology.

Moreover, as LRS technologies continue to advance, the integration of these methods into routine clinical practice is expected to enhance the precision of genetic diagnostics. This integration will not only improve the identification of pathogenic variants but also facilitate the exploration of the interactions between genetic and environmental factors in disease development (Warburton & Sebra, 2023) [5].

In summary, the applications of long-read sequencing in personalized medicine are vast and multifaceted. By improving the accuracy and efficiency of genetic diagnostics, LRS holds the potential to revolutionize patient care through more personalized and targeted therapeutic strategies. Its ability to elucidate complex genomic structures and variations is essential for advancing our understanding of genetic diseases and enhancing the effectiveness of precision medicine.

7 Conclusion ​

Long-read sequencing technologies have revolutionized the field of genomics, offering unprecedented capabilities in the analysis of complex genomic regions and structural variations. The primary findings indicate that long-read sequencing excels in detecting structural variants, characterizing full-length transcripts, and elucidating epigenomic landscapes, which are often challenging for traditional short-read methods. The current state of research highlights the rapid evolution of these technologies, with significant advancements made by companies like Pacific Biosciences and Oxford Nanopore Technologies. As the cost of long-read sequencing continues to decrease and its accuracy improves, its applications in clinical settings are expanding, particularly in personalized medicine and rare disease diagnostics. Future research directions should focus on enhancing computational tools for data analysis, integrating long-read sequencing into routine clinical workflows, and exploring its potential in population-scale studies. Overall, long-read sequencing holds the promise to further transform our understanding of genetic diversity and disease mechanisms, paving the way for advancements in precision medicine and targeted therapies.

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