New Inv. Award: Developing single-cell isoform sequencing tools to explore the diversity and regulation of alternative splicing in haematopoiesis

Lead Research Organisation: Earlham Institute
Department Name: Research Faculty

Abstract

The cell is a fundamental unit of biology - all multicellular organisms consist of populations of billions, even trillions of cells - many of which will have differing functions within the organism. The diversity of cell function arises from the ability of the cell to regulate and orchestrate the repertoire of genes those cells express.

One key mechanism cells use to increase the complexity of this repertoire is a process called alternative splicing. This is a regulated process where, during the process of gene expression, genetic information can be selectively excised from messenger RNA molecules. This can result in the generation of multiple protein variants from a single gene, and often these variants can have functionally distinct roles in the cell. It is by this means that the functional complexity of over 20,000 protein coding genes in the human genome can be increased by a factor of 5-10 - so from a relatively small number of genes, a larger variety of gene expression and function is possible.

Recent advances in DNA sequencing technology have enabled researchers to study the genetic information - RNA, DNA and epigenetic modifications to the DNA - contained within single cells. This has allowed a totally new perspective on the complexity and diversity of cell types that make up an organism. These techniques are broadly applicable to different organisms, and in human health and disease. However, to date, little has been done to explore the nature of alternative splicing in single cells, in spite of the important role this process plays in normal development of plants, animals and humans, and indeed in human diseases such as cancer.

In this proposal, we seek to generate new approaches that will reveal not just the extent of alternative splicing in single cells, and small populations of cells, but give parallel insight into the regulation of this process. While the methods we develop could be applied to almost any multicellular organism, we will use the technique to explore these processes in the development of normal blood cells in the mouse.

We have previously developed methods for parallel analysis of the genomes and transcriptomes of single cells, and further developed these methods to include epigenetic information - in the form of DNA methylation. By expanding these methods to work with so-called "long read" sequencing technology, we will create a platform which allows us to read out the full complement of splicing variation in individual cells. In parallel we will be able to explore how alternative splicing might be regulated by DNA methylation.

By using normal blood cell development as a testing ground for this new technology, we will reveal for the first time the amount of variation in alternative splicing in small populations of cells and single cells for which the function is very well understood. This information will be useful in enhancing our understanding of how blood stem cells make decisions, and how this complex system can sustain the generation of billions of new cells every day. Furthermore, by looking at cells from young and aged mice we will examine how the use of alternative splicing changes with age in these cells.

Technical Summary

Alternative splicing (AS) is a fundamental mechanism by which cells can generate diversity from a limited number for protein coding genes. Here, we seek to develop and apply tools which enable the analysis of AS at single cell resolution. Furthermore, we aim to integrate these observations with epigenetic measurements from the same single cell, to enable, for the first time, investigation of the epigenetic regulation of AS. Using Pacific Biosciences "long-read" sequencing technology, we will generate end-to-end sequencing reads from individual cDNA molecules from single cells. We will incorporate both cellular barcoding and unique molecular identifiers, such that samples can be multiplexed where possible, and that individual molecules can be identified and PCR duplicates can be excluded. We will further adapt existing informatics tools to enable comprehensive analysis of this data.

The project consists of an initial development phase, in which the technology will be optimised using well characterised cell-line models. We have generated preliminary data which indicates that the single cell transcriptome sequencing methods developed by the PI are compatible with long-read sequencers; in this phase we will improve the methods used to generate this preliminary data to generate a robust pipeline which will enable full-length isoform sequencing from single cells, and small populations of cells. In the subsequent application phase, the technology will be applied to small populations (50 cells) and single cells from the mouse haematopoietic system. These cells, including stem and progenitor cells, are functionally well characterised; however there is currently little understanding of the diversity and regulation of AS in these cells. By applying long read isoform sequencing, in parallel with DNA methylation analysis of the same single cell, we will, for the first time, enable exploration of these aspects of one of the best understood models of stem cell function.

Planned Impact

Academic Impact

The development of tools which enable study of alternative splicing and its regulation at the single cell level will be of direct benefit to academics working in genomics research, basic and haematological research as well as translational or clinical research.

Understanding the regulation of gene expression is of fundamental importance in biology, and we will generate data which will explore, for the first time, the epigenetic regulation of alternative splicing at the level of single cells and small pools of cells. Through analysis of the mouse hematopoietic system, we will generate data from a well characterised model of stem cell maintenance and differentiation which will act as a resource for genomic and non-genomic scientists working in haematological fields.

Training and career development

The research programme includes the training of a post-doctoral research associate, who will gain expertise in multi-disciplinary research, including cell biology, single cell isolation and sequencing and bioinformatics.
Furthermore, the methodology proposed in the research programme can be applied across a broad range of biological fields, and it is anticipated that, through release of protocols (publications, lab visits and workshops) we will enable researchers working on diverse model systems to readily apply the techniques.

Societal and Economic Impact

Single cell genomics is a rapidly growing field, and has driven the development of many technical developments in next generation sequencing, including the concept of "multi-omics", where more than one analyte (DNA/RNA/Protein) is analysed from the same single cell. We will liaise with the Knowledge Exchange and Commercialisation (KEC) team at EI to ensure that opportunities for economic impact, in particular interaction with industry, are fully exploited. This may include establishment of iCase studentships with commercial partners or the provision of specialist consultancy to companies which wish to apply or commercialise any methods arising.

We will also endeavour to communicate the findings of the work with the general public, either through blogging or providing case studies on the EI website or participating in outreach events.
 
Description This project, which commenced in October 2017, has already developed experimental approaches for sequencing the entire length of RNA molecules derived from individual cells. In the next year(s) we will further improve the methods and implement them to study primary biological systems including mouse blood cell development.
Exploitation Route We believe these techniques, once refined, will be widely applicable across many aspects of cellular genomics, including plant, animal and biomedical research.
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

 
Description Can there be only one? Exploring heterogeneity in RNA splicing at single-cell resolution
Amount £60,000 (GBP)
Funding ID 2117010 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2018 
End 09/2022
 
Description Development of single-cell sequencing technology for microbial populations
Amount £149,610 (GBP)
Funding ID BB/R022526/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2018 
End 06/2019
 
Description NRP Seed fund
Amount £16,000 (GBP)
Organisation Norwich Research Park 
Sector Private
Country United Kingdom
Start 01/2018 
End 01/2019
 
Description Single-cell cancer evolution in the clinic - zero rating
Amount £2,024,295 (GBP)
Funding ID C309/A26815 
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 10/2023
 
Title Single Cell Genomics at EI 
Description We have introduced single-cell genomics approaches to the institute. This includes development of facilities for tissue culture/processing, cell sorting (FACS) and automated pipelines for single cell RNA (Smart-seq2), DNA (MDA and Picoplex) and parallel genome and transcriptome analysis (G&T-seq). 
Type Of Material Improvements to research infrastructure 
Year Produced 2017 
Provided To Others? No  
Impact This has enabled the institute to interact with researchers across the campus to develop new avenues of research in plant, animal and microbial research. The relevant technologies have already supported the New Investigator Award associated with this outcome, as well as the funding of an NRP seed fund award as well as introduced several new collaborations to the institute. 
 
Description Haem - Cambridge 
Organisation University of Cambridge
Department Department of Haematology
Country United Kingdom 
Sector Academic/University 
PI Contribution We are working on technology development for single cell sequencing, using blood cells as a model system. In this collaboration we work with researchers at the Department of Hematology at Cambridge to ensure the technology development is in line with the state-of-the-art in blood biology
Collaborator Contribution At the moment, the contribution is in the form of intellectual interaction but will soon include the receipt of primary mouse cells and human cell lines which will support the development of the project.
Impact none as of yet
Start Year 2018
 
Description Flexible Talent Mobility Account Launch Workshop - 21 March 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact This workshop brought together ten (10) industry representatives from seven (7) different companies, ranging from Information Technology/Hardware, to transnational consumer goods companies, breeders and Research and Development companies with some of our project leaders to discuss potential areas for collaboration and skills transfer. We have since awarded seed funding to initiate pilot projects engaging EI faculty with companies in attendance for this workshop. In addition a number of NDAs are under development.
Year(s) Of Engagement Activity 2018
URL http://www.earlham.ac.uk/flexible-talent-mobility-account-and-workshop
 
Description Flow Cytometry Workshop 26 February 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact This training was a one-day lecture/theory-based workshop aimed at those who have access to Flow Cytometers and were in the planning stages of a project which would require their use. The two trainers were external experts - Dr Rachael Walker, running the Babraham Institute facility; Dr Derek Davies, running the facility and training/outreach associated at the Crick Institute. Aimed at beginners in the early planning stages of their experiments, the 21 attendees comprised mainly PhD students (~38%) and PostDocs (~28.5%) with other facility/service providers also in attendance. Feedback is still be collected and reviewed at the time of reporting.
Year(s) Of Engagement Activity 2019
URL http://www.earlham.ac.uk/flow-cytometry-training
 
Description Lego DNA Sequencing - Blocksford Brickopore 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Dr Iain Macaulay and Dr Richard Legget build a LEGO DNA sequencer using LEGO Mindstorms, which was displayed for use at the EI stand at the Norwich Science Festival. The instrument allowed users to "build" a 22-mer sequencer of DNA from 4 different coloured "bases" and then sequence them. Assistants from EI were then able to assist with blasting sequences and telling users what species their sequence aligned to. It was well received and we will use it agin at other outreach opportunities. The instrument even has its own twitter account (@brickopore)
Year(s) Of Engagement Activity 2018
URL http://twitter.com/@brickopore
 
Description Meiosis and Beyond - 5-6 March 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact A mini symposium bringing together researchers from a wide variety of fields united by one common theme - meiosis (before, during and after). Delegates and presenters working on a variety of organisms from plants to birds to nematodes came together to hear some of the latest research using new and emerging technologies and approaches in the field of meiosis. Beyond the mini symposium the group gathered for discussions including challenges they face in common with one another, technology that could help to answer their biological questions and to roadmap where they were as a research cohort.

The group are working on a paper summarising the current state and challenges and a subset have taken discussions from the workshop to successfully apply for funding to support new and emerging ideas.
Year(s) Of Engagement Activity 2018
URL http://www.earlham.ac.uk/meiosis-and-beyond
 
Description Norwich Single Cell Symposium 16-17 May 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The symposium was predominantly aimed at researchers across the region and attracted some 77 people with international speakers plus sponsors to discuss the latest development and challenges in the field. Offering sponsorship opportunities meant additional support for costs for the symposium but also a high degree of engagement from these sponsor companies, who were interested in our applications of Single Cell Technologies.
Year(s) Of Engagement Activity 2017,2018
URL http://www.earlham.ac.uk/norwich-single-cell-symposium-2018
 
Description Norwich Single-Cell Symposium - 8 May 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact With the developments in single-cell genomics technologies, particularly in the broadening of applications, we organised this symposium to bring together researchers who were curious about single-cell genomics with external speakers engaged in advanced single-cell research as well as highlighting the single-cell capabilities available at Earlham Institute. The idea was that this would provide an informal platform to catalyse future development and application of single-cell genomics across the region. The one-day symposium covered single-cell genomics topics in the following areas: technology development, developmental biology, health and disease, plant science and data analysis. The inaugural event had over 90 registrations, including a small number of industry representatives who had the option to sponsor and exhibit at the meeting. Feedback received suggested that the symposium offered a great diversity of topics in an informal and friendly setting. Many respondents providing feedback commented upon the high standard of presentations and enjoyable networking opportunities citing that they would like to see a repeat symposium the following year and that the meeting should be a little longer, with more networking.
Year(s) Of Engagement Activity 2017
URL http://www.earlham.ac.uk/norwich-single-cell-symposium
 
Description Single-cell RNAseq Training Course Delivered 3-7 Dec 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was the first iteration of this course, demonstrating EI's both introducing Single Cell Genomics and demonstrating EI's capability. It covered several aspects such as: the experimental design, cell sorting and processing for production of quality samples for sequencing, in the three-days of laboratory hands-on sessions; generation of sequencing data, assessing the quality of sequence data, data visualisation, differential expression analyses and identifying Copy Number Variants at the single cell level in the following two-days of theory and computational hands-on sessions. A total of twelve (12) delegates attended this course, with ten (10) attending the full five days. Also included was a short half-day session with practical elements on single molecule FISH, delivered by EI faculty, and an overview of 10X Genomics' data analysis platform, presented by one of their Field Application Specialists.

Delegates were predominantly UK-based (~83%) and PostDocs (two-thirds), with a couple of attendees responsible for similar laboratory facilities in their home institutions. All attendees rated the trainers as "Very Good" to "Excellent", and a survey of the ten laboratory sessions attendees suggested that, having followed the course, many hoped to engage with a Single-Cell RNAseq service for their research (53% very likely and 40% likely), with 53% wishing to provide cells.

Following the unprecedented success of delegates producing high quality data to analyse, course organisers wish to review the programme. The proposal for the second iteration is to run the course as 3 or 4 days in the laboratory, with sequencing running over a weekend, and then extending the bioinformatics from 2 days to 4 days, running this on the following week.
Year(s) Of Engagement Activity 2018
URL http://www.earlham.ac.uk/single-cell-rnaseq-training-course
 
Description Training - Single-Cell RNAseq Training Course 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Training delivered as part of the Single-Cell RNAseq Training Course at EI. The audience included postdoctoral researchers and graduate students
Year(s) Of Engagement Activity 2018