Our researchers are combining single cell approaches with long read sequencing technologies, unearthing data that reveals the scale and impact of cellular diversity.
Single-cell and spatial technologies continue to develop at pace. Their emergence promises fresh insights into biology and a transformed picture of the complexity of life.
New platforms and approaches are helping even recent converts to investigate hitherto unknown cellular diversity.
Despite the excitement, research groups relying solely on short read data have only been able to see a fragment of the larger picture.
“When you do single cell RNAseq with short reads, for example, you lose loads of crucial information,” explains Dr Iain Macaulay, a Group Leader at the Earlham Institute with more than a decade of expertise in single cell research.
“In my group, one of the things we want to understand is the role of alternative splicing events on cell differentiation.
“There’s evidence that alternative splicing plays a role in the haematopoietic system, so we know it’s an area we should be exploring.
“But we’d completely miss the information we need if we only had access to short read technologies.”
Investigating the functional impact of cellular variation - both in health and disease - is an emerging area of research, and a core part of the Earlham Institute’s strategic research programme, Cellular Genomics.
The latest single cell platforms and approaches are needed to tackle these questions.
“Thanks to long-term investment from BBSRC, the Earlham Institute’s capability has gone from a sole instrument to a dedicated single cell lab and research group,” says Iain.
Unlocking their full potential, however, has hinged on the maturation of another technology the Institute has a long association with.
“Long read sequencing technologies have really arrived now, which means we’re able to use them in our single cell research,” adds Iain.
Long-read sequencing technologies are now increasingly widespread, affordable, and being innovatively applied to a wide range of research questions.
Their marriage with single cell approaches is something the Earlham Institute has pioneered, with Iain’s group particularly interested in understanding the role of alternative splicing on cell differentiation.
“We’ve been trying to take this kind of approach for nearly 10 years now, having first applied long-read sequencing to single cells during the development of the G&T-seq protocol,” says Iain.
“That’s largely because of the increased throughput and fall in costs of long read technologies, along with the development of methods that link single-cell approaches with long-read library preparation.”
“We’ve also seen a huge expansion of single cell approaches and far greater awareness out there of the field’s value. This combination is helping us to build the expertise - and secure the funding - to ask some really exciting questions.”
When you’re innovating with these new instruments and approaches, a lot of time and energy is spent in technical development. We’ve now reached a point where we can turn up the volume on the biology.
Dr Iain Macaulay (right) during a Cellular Genomics programme meeting.
Within Iain’s research group are a number of projects combining single-cell and long-read technologies, with a particular focus on cell differentiation and immunology.
“When you’re innovating with these new instruments and approaches, a lot of time and energy is spent in technical development,” explains Iain. “We’ve now reached a point where we can turn up the volume on the biology.”
Dr Anita Scoones, a postdoc in the Macaulay Group, has been involved in some of the technical development underpinning long read single cell sequencing.
Anita is applying her approaches to blood cell differentiation, identifying which isoforms of genes are expressed as cell fate is chosen.
Charlotte Utting, a PhD student in the group, is perturbing gene expression and looking at the impact on alternative splicing during cell differentiation.
Her project builds on the work Anita has been doing. But she has also been driving her own ‘tech dev’ with crispr technologies for her perturbation experiments.
Charlotte’s work, which may be the first of its kind, will bring these technologies and approaches together to explore the role of RNA binding proteins in haematopoiesis.
Senior Research Assistant Ashleigh Lister (left) with PhD researcher Charlotte Utting (right)
Also in the Macaulay group is Dr Meha Patel, a Wellcome Clinical PhD Fellow. She is using single cell technologies to define the tissue-resident and circulating immune cell landscape in Primary Sclerosing Cholangitis (PSC), a rare liver disease.
“We know very little about PSC,” explains Meha, “but the health burden - in the UK and globally - is significant despite its rarity. Just over one in ten liver transplant operations in the UK are due to PSC.
“Previous research has pointed to a role played by T-cells, so I’m looking specifically at immune cells from patients with suspected and confirmed PSC, along with other auto-immune diseases.”
Not all patients with PSC will undergo a procedure where the tissue needed for this project can be collected, therefore maximising the information gained from each precious sample is crucial.
Short read technologies would only capture one isoform. Long reads are needed to reveal detail that would otherwise be lost about the activity of these immune cells, while single cell approaches capture the heterogeneity across a tissue sample.
“Ultimately, we want to use the combination of these two technologies to identify potential targets for therapy,” says Meha.
“We want to transform the understanding of PSC and the prognosis for patients. Every tissue sample we receive is hugely valuable so I feel we have a duty to really maximise the useful information we can gather from them.”
Meha’s project is one of the first within the Macaulay Group to direct this technology development and multidisciplinary research towards clinical applications.
“Meha is looking at immune cells in the context of disease,” says Iain, “and single cell techniques let us access these cells.
“We can then use long reads to help us explore things like immune receptor specificity.”
Both Anita’s and Charlotte’s projects are part of the Cellular Genomics work package ‘Cell expression heterogeneity impact on environmental response’.
The work package is linking heterogeneity in gene expression within individual cells with whole organism phenotypes and adaptability.
Ultimately, we want to use the combination of these two technologies to identify potential targets for therapy.
Researchers Dr Anita Scoones and Dr David Wright (standing) during a single-cell RNAseq training course held at the Earlham Institute
At the Earlham Institute, both long-read technologies and single cell approaches are employed across both of its strategic research programmes - Cellular Genomics and Decoding Biodiversity.
The former is investigating genome variation and regulation while the latter is employing them to explore complex microbiomes.
The UK bioscience community can access the Institute’s platforms in these and other technologies through its BBSRC-funded National Bioscience Research Infrastructure (NBRI) - Transformative Genomics.
The Transformative Genomics programme brings together technical expertise, equipment, and facilities to engage with and exploit emerging technologies for genome science, with a particular focus on long-read sequencing, single-cell and spatial analysis, and genomics at scale
Within the team is a wealth of expertise in a range of next-generation sequencing platforms and workflows, allowing them to tailor each project to the user’s needs.
And the Institute’s Advanced Scientific Training team coordinates a calendar of events and courses for you to develop your knowledge or discover the power of these technologies.
