The Human Genome Project was a historic milestone, but it only scratched the surface. While it delivered the first working draft of the human genome, roughly 8% remained unresolved. Most of these gaps fell within the noncoding regions that make up over 90% of our DNA¹. These difficult-to-sequence areas, known as the “dark genome”, are dense with repeats, rich in GC content, and layered with epigenetic complexity, making them especially challenging for traditional short-read methods to resolve.
Once dismissed as non-functional “junk DNA,” the dark genome is now understood to be biologically meaningful. It houses regulatory elements, structural variants, and other features that play critical roles in gene expression, development, and disease biology²˒³.This article explores how long-read sequencing (LRS) is helping researchers access these once-intractable regions, uncovering actionable variants and epigenetic signatures that short-read methods often miss.
Short-read sequencing (SRS) has powered genomic discovery for decades, but its design poses inherent limitations when analyzing complex genomic regions. The method requires DNA fragmentation and PCR amplification prior to sequencing, which disrupts long-range sequence context, introduces amplification bias, and eliminates base-level epigenetic information4-6.
These limitations are especially problematic in dark regions enriched with tandem repeats, high GC content, and large structural variants such as deletions, insertions, and translocations7,8. These features complicate alignment and variant calling, even with advanced bioinformatics pipelines4. Additionally, profiling DNA methylation with SRS requires bisulfite conversion or immunoprecipitation, which can degrade input DNA, introduce artifacts, and prolong assay time 6,9. As a result, large portions of the genome, including potentially pathogenic variants and regulatory elements, remain inaccessible to standard sequencing workflows.
Long-read sequencing (LRS), including technologies such as Oxford Nanopore, offers a fundamentally different approach. By sequencing intact, native DNA or RNA strands without fragmentation or amplification, LRS preserves long-range structure and retains nucleotide modifications such as 5-methylcytosine and 5-hydroxymethylcytosine6,10,11.
These long native reads can span repetitive elements and GC-rich regions, enable base-resolution detection of structural variants, and simultaneously capture epigenetic modifications. Together, these advantages make LRS uniquely suited to interrogate the dark genome with greater precision and clinical relevance.
Despite the broad adoption of sequencing in clinical genetics, approximately 50% of suspected Mendelian conditions remain undiagnosed12. Many of these unresolved cases involve variants in regions poorly covered, poorly mapped, or structurally inaccessible to SRS.
LRS has emerged as a valuable tool for resolving these diagnostic blind spots. Studies have shown its ability to detect short tandem repeat expansions13, intronic and regulatory region mutations3, and structural anomalies like deletions or translocations2,3. In neuromuscular syndromes, Gitelman syndrome, and other conditions, long-read methods have led to pathogenic variant discovery that would not have been possible with conventional short-read sequencing2,3,13.
Tumor genomes often exhibit extensive structural remodeling, including gene fusions, amplifications, and chromosomal rearrangements, that are difficult to resolve with SRS. Long-read sequencing enables comprehensive structural variant detection, even in repetitive or GC-rich regions. For example, in HER2+ breast cancer and pancreatic tumor models, Oxford Nanopore sequencing has identified tens of thousands of structural variants, the majority of which were missed by short-read approaches14-16. These included interstitial deletions, inversions, translocations, and complex fusion events, often resolved at moderate coverage with a fraction of the reads required by SRS16.
LRS also provides integrated methylation data from native DNA, facilitating tumor classification. In CNS tumors, nanopore-based methylation profiling reproducibly classified more than 80 tumor subtypes, often delivering results faster and with fewer preprocessing steps than bisulfite-based workflows17,18. This dual-resolution capability—genomic and epigenomic—positions LRS as a compelling diagnostic modality in cancer research and clinical application.
The value of long-read sequencing lies in both its molecular resolution and operational efficiency. By sequencing full-length, unamplified DNA or RNA:
Large structural variants and repetitive elements are more reliably detected
Methylation and sequence data can be captured from a single assay10
Turnaround times and sample input requirements are reduced
Lower sequencing depth is required for clinically meaningful results16
For translational researchers, diagnostic developers, and precision medicine teams, LRS represents a scalable platform for high-yield discovery in previously intractable regions of the genome.
Once dismissed as “junk DNA,” the dark genome is increasingly recognized as a source of biologically and clinically relevant information. Long-read sequencing provides the necessary resolution to interrogate these regions with greater confidence and context.
As long-read platforms like Oxford Nanopore continue advancing, their ability to simultaneously capture sequence and methylation information in native molecules may enable broader and more integrated analyses of the genome. For researchers and clinical teams, the dark genome is no longer a technical blind spot, it’s an opportunity to understand complex disease mechanisms and refine molecular diagnostics.
Figure created with BioRender
