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

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.

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.

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.