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

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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.