Single-Cell Transcriptomics Project Will Offer New Understanding of the Immune System
A new, collaborative project will harness long-read single-cell RNA sequencing to explore gene activity in the human immune system.
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Advances in next-generation sequencing (NGS) technologies have lowered costs and reduced the complexity of DNA and RNA sequencing, enabling new possibilities when studying gene activity. In collaboration with Pacific Biosciences, scientists at the Wellcome Sanger Institute will harness these advancements to explore how gene activity shapes the human immune system at single-cell resolution.
Technology Networks had the pleasure of interviewing Dr. Gosia Trynka, group leader at the Wellcome Sanger Institute and science director of Open Targets, and Dr. Elizabeth Tseng, associate director at PacBio, to explore the evolution of single-cell transcriptomics, its utility for studying human disease and the aims of the new project.
Single-cell transcriptomics revolution
There are two main NGS methods used in modern research: short-read and long-read sequencing. This major new research initiative marks the first time that the Sanger Institute will use long-read single-cell RNA sequencing at scale.
Trynka explained how long-read sequencing differs from short-read sequencing: “Long-read single-cell RNA sequencing is an emerging technology that allows us to capture the full-length transcripts of RNA molecules from individual cells. This is a step-change from traditional short-read sequencing, which typically reads RNA fragments in small pieces that must be computationally stitched together.”
“This is important because genes often produce multiple transcript variants, so-called isoforms, through a process called alternative splicing. With short reads, we only get a partial view that misses this complexity. Short-read sequencing allows us to estimate the total abundance of transcript molecules without specifying which isoforms they come from. Long-read sequencing lets us see exactly which isoforms are present, and when applied at a single-cell level, it gives a very granular view of transcript complexity in each cell type. In short: combining long reads with single-cell resolution gives us a more complete and nuanced picture of gene activity, one that’s closer to the biology itself,” Trynka added.
This capability is especially valuable when studying complex systems, such as the immune system or diseases like Inflammatory Bowel Disease (IBD), where small variations in RNA processing can lead to significant differences in cell behavior during immune challenges or in response to treatment.
The fundamental role of RNA isoforms in gene regulation hasn’t always been appreciated. Historically, many studies focused on whether a gene was turned “on” or “off” and quantifying its expression. Tseng noted an emerging consensus that RNA splicing plays a critical role in all areas of disease research: “For example, de novo mutations in the genome that lead to aberrant splicing can be cause for genetic rare diseases. Certain types of cancer might have widespread mis-splicing patterns.”
“Some isoforms are more dominant than others in specific cell types in the brain that are associated with neurological diseases. Understanding RNA splicing for different diseases can lead to better therapeutics,” she added.
“We now know that most human genes produce multiple isoforms, which can lead to proteins with very different, and sometimes opposing functions. These differences can affect everything from how immune cells respond to infection, to how a person reacts to a drug,” said Trynka, adding that a classic example is CD45, a key surface molecule found on T cells. “The CD45 gene produces multiple isoforms through alternative splicing, notably CD45RA and CD45RO. These isoforms are not interchangeable, they mark functionally distinct T cell states: CD45RA is predominantly expressed on naive T cells, which have not yet encountered antigen – an immune challenge. CD45RO is expressed on memory T cells, which have previously responded to a pathogen and can mount a faster, more robust immune response,” she said. “This isoform switch plays a critical role in how the immune system remembers and responds to threats. And it illustrates how isoform diversity can serve as a marker and a mechanism of cellular function.”
However, a clear, systematic understanding of how transcripts can vary between cell types and people, and how gene variants regulate these alternative splicing events, is lacking.
“Uncovering this layer of gene regulation is essential for linking genetic risk to cellular function, particularly in complex diseases like IBD and beyond,” Trynka said.
Applying long-read single-cell transcriptomic sequencing at scale
In the new project, Sanger Institute researchers will apply PacBio HiFi sequencing to ~1,500 blood and gut tissue samples from several ongoing clinical studies: IBDverse, IBD-Response and JAGUAR.
IBDverse and IBD-Response are investigating novel ways to treat IBD. JAGUAR, in contrast, is a large-scale study across seven Latin American countries that is exploring how genetic diversity impacts or shapes our immune response. The new project will create high-resolution maps of both RNA expression and splicing across different cells, tissues and people.
“These studies were a natural fit because they bring together three essential ingredients for this kind of high-resolution transcriptomic work: clinical relevance, biological complexity and population diversity,” said Trynka. “IBDverse and IBD-Response are focused on a complex, immune-mediated disease – IBD – which affects millions worldwide but remains difficult to treat effectively. Many patients don’t respond to existing therapies and we suspect that hidden layers of gene regulation, including isoform usage and cell-type-specific expression patterns, may hold the key to understanding these differences. These cohorts already include rich clinical data and tissue samples from the gut – the site of disease – making them ideal for investigating how gene activity and splicing vary in the context of IBD.”
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“JAGUAR, on the other hand, brings something equally critical: ancestral and geographic diversity. It focuses on how genetic variation across Latin American populations influences immune responses. These populations have been vastly underrepresented in genomics research, yet they offer enormous opportunities to discover novel regulatory mechanisms that might be absent or rare in European-centric studies. By including samples from Peru and Mexico, we can explore how isoform usage is shaped not only by cell type but also by genetic background,” Trynka added.
The new project will create high-resolution maps of both RNA expression and splicing across different cells, tissues and people. Credit: iStock.
A new chapter for single-cell transcriptomics
The project leaders are deeply committed to open science. All results from the project will be published in peer-reviewed journals and will be accompanied by public data releases. “Our data releases will align with major publications, expected over the next 12–24 months, depending on the maturity of each analysis. In addition, we aim to release intermediate resources, like splicing maps, isoform annotations and computational tools as early as possible to accelerate progress across the field,” said Trynka.
Genetic and transcriptomic datasets will be shared via secure, managed-access platforms such as the European Genome-phenome Archive to protect participant privacy. “This allows researchers to access the data while maintaining protections for sensitive genetic information,” Trynka added.
For the Wellcome Sanger Institute, this project forms part of a broader effort to transition away from cataloging the genome and towards building an understanding of how it works, particularly across diverse populations and varied diseases.
“By generating isoform-resolved, single-cell data at scale, we hope to reveal new layers of gene regulation that have so far been invisible to standard methods, and to make those insights widely accessible through open data sharing and new computational tools,” Trynka said. By partnering with PacBio, the Institute aims to demonstrate how high-fidelity long-read sequencing technology can “transform transcriptomics”, she added: “This project is also an important step in making these advanced methods more scalable, reproducible and usable across large cohorts and population studies.”
For PacBio, participating in Sanger’s large cohort studies will help make new datasets available to the scientific community, which may drive new insights, Tseng said.
“Together, we’re not just building a dataset, we’re laying the groundwork for a new chapter of transcriptomics. Ultimately, we want to enable a better understanding of human biology that leads to better, more equitable healthcare solutions,” Trynka concluded.
