Rare diseases are uncommon on their own, but together they affect a surprisingly large number. More than 300 million people worldwide live with one of over 7,000 distinct conditions, most of which have a genetic basis. But for many families, the path to diagnosis is long and uncertain. Even after several rounds of genetic tests, including short-read sequencing, more than half of suspected rare disease cases remain unresolved.
In recent years, long-read whole-genome sequencing has galvanized overdue progress in rare disease research. By preserving long stretches of native DNA, this technique can resolve complex structural variants, repeat expansions, paralogous genes, and other difficult regions that conventional testing methods frequently miss. As a result, long-reads have delivered meaningful gains in diagnostic yield, providing answers for patients who were previously thought to have reached the end of the diagnostic road.
Yet the diagnostic odyssey still impacts many patients, placing a heavy burden on families, clinicians, and healthcare systems alike. This persistent gap underscores a central challenge in modern pathology: biological complexity often extends beyond what can be read from DNA sequence alone.
Rare diseases are not defined by the genome in isolation. The same genetic sequence can lead to very different biological outcomes depending on how genes are regulated, expressed, and packaged within the cell. To unlock the next wave of progress in rare disease research, pathologists must increasingly look beyond the genome and adopt a multi-omic view of disease biology.
Introducing the four 'omes
There are four biological layers proven to be relevant to rare disease biology: the genome, transcriptome, methylome, and chromatin epigenome. Together, these layers provide a more complete picture of how genetic variation translates into characteristics or symptoms.
The genome
The genome is well understood by pathologists. In short, it represents the blueprint of biology that encodes instructions for bodily development and function. The genome has traditionally been the primary focus of genetic testing. Long-read sequencing has significantly improved researchers’ ability to interrogate the genome, particularly in complex regions linked to rare disease. However, the genome describes potential rather than outcome. Alone, it does not explain how or when genes are used, meaning scientists cannot determine when, or how severely, conditions might develop.
The transcriptome
For the genome’s instructions to be carried out and proteins to be made, DNA must first be "read" and copied onto RNA molecules. These readouts are the transcriptome, and the RNA transcripts determine which proteins are produced, in what quantities, and in which tissues. In rare disease, disruptions at the transcript level, such as aberrant splicing or altered expression, can have profound functional consequences even when the underlying DNA sequence appears unchanged.
The methylome
The methylome reflects chemical modifications to DNA, most notably the addition of methyl groups to cytosine bases. These epigenetic marks do not alter the genetic code, but influence whether genes are switched on or off. Abnormal methylation patterns can silence critical genes or disrupt developmental pathways, contributing to disease mechanisms that remain invisible to genome-only analyses.
For example, a methyl group might "silence" tumor suppressor genes, which normally act to prevent cancer by controlling cell growth and repairing DNA damage. Since methyl groups do not directly change the DNA strand, it would be challenging to spot signs of cancer by only looking at the genome until physical tumors formed.
The chromatin epigenome
DNA is packaged within the cell as chromatin, a highly dynamic structure that determines how accessible different genomic regions are to the cellular machinery. Changes in chromatin organization can either restrict or enable gene expression by altering how tightly DNA is wrapped. In rare disease, disruptions to chromatin structure can misregulate entire gene networks, affecting biological function without changing the DNA sequence itself.
A fragmented approach with limited insights
Viewed in isolation, each 'ome provides only a partial perspective on rare disease pathogenicity. Until recently, this fragmented view was largely unavoidable without imposing significant additional time, cost, and testing burden on patients. Analyzing multiple 'omes required several assays, technologies, and datasets, often generated separately and interpreted in isolation.
Genomic analysis has also traditionally focused on coding variants – changes within genes that alter the structure of a protein and are therefore easier to interpret. However, these account for only a small fraction of genetic variation. Ninety-eight percent of variants lie in non-coding regions of the genome, where they can influence when, where, and how genes are regulated, often in ways that are far harder to predict. Increasingly, it is thought that the underlying cause of many rare diseases lies within this vast non-coding landscape that is still poorly annotated.
Technology advances: one test for four 'omes
Now, advances in next-generation multi-omic sequencing are enabling a shift away from this fragmented model. Highly accurate long-read approaches, such as HiFi sequencing, make it possible to analyze DNA and RNA to interrogate multiple 'omes within a single, integrated workflow. This consolidation also reduces reliance on multiple legacy assays, saving time and preserving precious patient samples.
Additionally, advanced multi-omics methods allow both coding and non-coding genetic variants to be linked to downstream effects on gene expression and chromatin organization. The ability to capture genetic variation together with its regulatory and epigenetic context is essential for understanding how non-coding variants contribute to disease mechanisms.
Progress has also been driven by collaboration across the sequencing and reagent ecosystem. Partnerships between sequencing platform developers and epigenomics specialists are maturing and scaling multi-omic workflows, combining advances in chemistry, library preparation, and analysis. For example, PacBio’s collaboration with EpiCypher intends to support more reproducible chromatin and methylation profiling alongside long-read sequencing.
Case study: rare disease research in practice
Multi-omic sequencing has the potential to bridge a long-standing divide between discovery research and clinical investigation. A clear example demonstrating the value of this integrated approach comes from researchers at the University of Washington. The team applied simultaneous long-read genome, methylome, chromatin, and transcriptome analysis to investigate a previously undiagnosed pediatric case.
The patient presented a complex constellation of symptoms, including developmental delay and bilateral retinoblastomas, which could not be explained by genomic sequencing alone. By examining all four 'omes together, the researchers were able to disentangle multiple disease mechanisms operating in parallel.
In this case, the patient’s developmental delay could be identified through the genome, caused by NBEA haploinsufficiency – when one of the two NBEA genes is missing or faulty. However, her bilateral retinoblastomas were caused by chromatin epigenetic change, specifically the deactivation of the RB1 cancer suppression gene. Since the change did not impact the DNA sequence, it could not have been identified by only analyzing the genome.
Moving rare disease pathology beyond the genome
For pathologists, this work illustrates how multi-omic sequencing can shift rare disease investigation from a process of elimination to one of biological explanation. By integrating multi-omic insight, pathologists can move beyond asking whether a variant exists to understanding how it disrupts biological function – an essential step in resolving complex and previously unexplained cases.
Looking ahead, the next wave of progress in rare disease research will depend on moving beyond genome-only approaches to routinely considering regulation, expression, and chromatin context. Realizing this shift will require sustained investment in rare disease research, collaborative infrastructure, and technologies capable of capturing biology in its full complexity.
Multi-omic approaches, particularly when paired with long-read sequencing, offer a way to link this non-coding variation to its downstream effects on gene expression and chromatin organization.
Newsletters
Receive the latest pathologist news, personalities, education, and career development – weekly to your inbox.
