Long-Read Sequencing: Powering High-Resolution, Multiomic Insights

1 Long-Read Sequencing: Powering High-Resolution, Multiomic Insights Katie Minns, PhD Long-read sequencing (LRS) is a powerful DNA and RNA sequencing technology that reads contiguous fragments routinely exceeding tens of kilobases, and in some cases over one million bases. This is dramatically longer than the hundreds of bases captured by traditional short-read sequencing (SRS) methods, which struggle with highly repetitive sequences and complex structural variants, creating regions of the genome that are invisible to diagnostics. LRS, however, can detect structural variants, single nucleotide variants, insertions, and deletions—capabilities that are revolutionizing genomics, transcriptomics, and epigenomics research, and driving its transition into clinical diagnostics and precision medicine.1,2 Leading platforms in the market include those using polymerase kinetics and current disruption technologies. For polymerase kinetics, large fragments of single circularized DNA molecules are sequenced in tiny wells, measuring fluorescent light emission during repeated passes with a polymerase to gain a high-accuracy consensus sequence. Current disruption LRS works by passing single DNA fragments through nanopores and measuring the disruption to the flow of ions, with algorithms determining the sequence.3 The LRS market is projected to grow from $1.15 billion in 2025 to $2.96 billion in 2029, driven by increased adoption in clinical diagnostics and precision medicine, expanded uptake in rare disease studies, and increased demand for comprehensive epigenetic profiling.4 This listicle will highlight the diverse applications of LRS, from resolving complex structural variants to detecting epigenetic modifications. It will showcase how these capabilities are advancing research in genomics, disease diagnostics, and precision medicine: from constructing complete reference genomes to enabling rapid diagnosis in critically ill patients. Applications of long-read sequencing High-quality genome assembly One of the most transformative impacts of LRS has been the assembly of complete, telomere-to-telomere genomes. Long reads can now traverse repetitive elements and highly complex regions that fragment short-read assemblies such as the current human reference genome (GRCh38). This has led to some of the most complete reference genomes yet. For example, the Telomere-to-Telomere (T2T) consortium has generated the first gapless human genome (T2T-CHM13).5 Complete reference genomes are essential to ensuring that all genomic variants are discovered and studied.6 Listicle LONG-READ SEQUENCING: POWERING HIGH-RESOLUTION, MULTIOMIC INSIGHTS 2 Structural variant detection LRS has revolutionized the analysis of structural variants. Large deletions, duplications, and inversions were practically invisible with SRS, which can’t resolve complexity in some regions. But now LRS can span an entire structural variant, including its breakpoints and surrounding sequence, providing clear evidence of the rearrangement. For researchers working on cancers such as esophageal or colorectal cancer, which have fusion genes, copy number aberrations, and other complex genome rearrangements, LRS is substantially more sensitive than SRS for detecting these events.7,8 Haplotype phasing Haplotype phasing determines which genetic variants are located together on a chromosome, which is essential for understanding inheritance patterns and allele-specific gene regulation. LRS can directly link variants along a single DNA molecule, producing high-accuracy haplotypes without requiring parental genotypes or statistical inference.7 A clinical example comes from full-length sequencing of the ABO blood group gene. Researchers used LRS to span from the 5′ untranslated region (UTR) to the 3′ UTR, generating complete ABO haplotypes. This approach resolved complex and rare ABO variants, including large fragment deletions, inversions, recombination, and chimeras, that are difficult to interpret with traditional genotyping, improving the understanding of blood group characterization for clinical use.9 Transcriptome profiling LRS enables complete transcriptome profiling by reading full-length RNA directly, rather than reconstructing transcripts from short fragments.7 This is particularly valuable for highly polymorphic genes, such as HLA genes, and recombinant genes, such as immunoglobulin genes, where short-read methods struggle to distinguish between similar transcripts due to repetitive sequences. The long-read approach captures the features that distinguish similar transcripts, such as polyadenylation sites and alternative exons. It also supports the discovery of novel isoforms, alternative splicing events, and fusion transcripts. Emerging applications of long-read RNA sequencing include single-cell transcriptomics, spatial transcriptomics, and discovering full-length isoforms as potential disease biomarkers.10 Epigenetic and epitranscriptome analysis A unique benefit of LRS platforms is their ability to automatically detect native DNA and RNA modifications, such as 5-methylcytosine and N6-methyladenine, without additional library preparation. Because single molecules are sequenced end-to-end, multiple epigenetic events can be measured on the same chromatin fiber, enabling co-association analyses and thorough epigenomic profiling. These epigenetic marks, associated with rare diseases or cancer, for example, are recognized as potential biomarkers that can support better research, diagnosis, and treatment.2 Chromatin architecture can also be identified by LRS assays, including nucleosome positioning, transcription factors, and chromatin accessibility regions along each read. This reveals which genes are accessible to be transcribed, and the impact of this on gene regulation. Listicle LONG-READ SEQUENCING: POWERING HIGH-RESOLUTION, MULTIOMIC INSIGHTS 3 Beyond DNA methylation, LRS-based epitranscriptomics reads full-length RNA molecules together with their chemical modifications, demonstrating how RNA methylation and related marks influence transcript stability, isoform usage, and cellular phenotype.2,7 Population genomics The T2T consortium’s gapless human genome is a huge achievement; however, using a single genome as a reference creates bias in variant detection. To overcome this, the Human Pangenome Reference Consortium has created a pangenome reference from a diverse group of 47 individuals. When applied to short-read data analysis, this pangenome increased the number of structural variants detected per haplotype by 104% compared with GRCh38-based workflows, uncovering previously hidden sources of genetic diversity.11 However, certain populations remain severely underrepresented in reference genomes and pangenomes.7 Studies such as the All of Us Research Program are beginning to tackle this challenge by creating a longitudinal cohort study that aims to enroll a diverse group of at least one million individuals across the USA to accelerate biomedical research and improve human health.12 Applications in clinical diagnostics and precision medicine LRS is transitioning into clinical diagnostic testing for genetic diseases, where a core advantage of the technology is that it can be used as a single test to replace multi-step workflows, accelerating the time to diagnosis. More than half of individuals with suspected rare diseases remain undiagnosed after genomic investigations, as SRS cannot reliably capture all types of variation.3 LRS addresses this gap by uncovering pathogenic variants missed with SRS, providing hope to those in need of a diagnosis.13 In one study, critically ill neonates and infants suspected of genetic disease were provided with LRS alongside standard genomic care. A comparison of diagnostic turnaround times showed an average of 5.3 days for LRS and 18.4 days for standard genomic care, showing that LRS enabled more rapid treatment decisions.14 In another study, targeted LRS performed on DNA extracted from blood replicated the results of standard newborn molecular screening for the rare disorders galactosemia and Pompe, while also phasing variants into haplotypes without the need for parental samples.15 For oncology patients, targeted LRS can be used to identify fusion genes, cancer-specific mutations, and biomarkers that guide precision therapy selection. To scale clinical use of LRS, standardized protocols and quality parameters must be established, along with innovative tools for data analysis and interpretation.3 From research tool to clinical standard LRS is a powerful advancement in genomic analysis, addressing gaps that have limited SRS technologies for decades. By reading through complex regions in single molecules and capturing full-length transcripts, LRS delivers unprecedented resolution across the genome, transcriptome, and epigenome—transforming research and clinical applications. Listicle LONG-READ SEQUENCING: POWERING HIGH-RESOLUTION, MULTIOMIC INSIGHTS 4 Tangible benefits have already been seen in clinical settings, with faster diagnoses for critically ill patients, improved detection of pathogenic variants in rare diseases, and more accurate characterization of cancer-specific mutations. As costs decline, diverse population databases expand, and bioinformatic workflows mature, LRS is set to become routine in clinical laboratories, embedding personalized medicine as a diagnostic standard. References 1. Logsdon GA, Vollger MR, Eichler EE. Long-read human genome sequencing and its applications. Nat Rev Genet. 2020;21,597–614. doi: 10.1038/s41576-020-0236-x 2. Liu T, Conesa A. Profiling the epigenome using long-read sequencing. Nat Genet. 2025;57,27–41. doi: 10.1038/s41588- 024-02038-5 3. Eisfeldt J, Ek M, Nordenskjöld M, et al. Toward clinical long-read genome sequencing for rare diseases. Nat Genet. 2025;57,1334–1343. doi: 10.1038/s41588-025-02160-y 4. Research and Markets. Long-read sequencing market report 2025. https://www.researchandmarkets.com/reports/6178253/long-read-sequencing-market-report. Published 2025. Accessed 15 January 2026. 5. Nurk S, Koren S, Rhie A, et al. The complete sequence of a human genome. Science. 2022;376,44–53. doi: 10.1126/science.abj6987 6. Miga KH, Koren S, Rhie A, et al. Telomere-to-telomere assembly of a complete human X chromosome. Nature. 2020;585,79–84. doi: 10.1038/s41586-020-2547-7 7. Marx V. Method of the year: long-read sequencing. Nat Methods. 2023;20,6–11. doi: 10.1038/s41592-022-01730-w 8. Xu L, Wang X, Lu X, et al. Long-read sequencing identifies novel structural variations in colorectal cancer. PLoS Genet. 2023;9(2):e1010514. doi: 10.1371/journal.pgen.1010514 9. Ying Y, Zhang J, Hong X, et al. Comprehensive annotation of complete ABO alleles and resolution of ABO variants by an improved full-length ABO haplotype sequencing. Clin Chem. 2025;71,(4),510–519. doi: 10.1093/clinchem/hvaf015 10. Monzó C, Liu T, Conesa A. Transcriptomics in the era of long-read sequencing. Nat Rev Genet. 2025;26,681–701. doi: 10.1038/s41576-025-00828-z 11. Liao WW, Asri M, Ebler J, et al. A draft human pangenome reference. Nature. 2023;617,312–324. doi: 10.1038/s41586- 023-05896-x 12. The All of Us Research Program Genomics Investigators. Genomic data in the All of Us Research Program. Nature. 2024;627,340–346. doi: 10.1038/s41586-023-06957-x 13. Sinha S, Rabea F, Ramaswamy S, et al. Long read sequencing enhances pathogenic and novel variation discovery in patients with rare diseases. Nat Commun. 2025;16(1):2500. doi: 10.1038/s41467-025-57695-9. 14. Smits DJ, Ferraro F, Drost M, et al. Nanopore long-read sequencing for the critically ill facilitates ultrarapid diagnostics and urgent clinical decision making. Eur J Hum Genet. 2026:34,108–118. doi: 10.1038/s41431-025-01959-x 15. Paschal C, Ye S, Myers C, et al. P693: Resolution of newborn screening results via targeted long-read sequencing. GIM Open. 2025;3,103062. doi: 10.1016/j.gimo.2025.103062 About the author: Dr. Katie Minns received her PhD in Biomedical Sciences in 2016. She developed her passion for molecular biology as a microbiologist in a contract research organization and as a healthcare scientist team leader at Public Health England. Katie moved into science communication in 2021, starting as an in-house scientific content writer at a life sciences company specializing in genomic analysis, and is now working on a freelance basis. Listicle