A 2018/2019 Summer Studentship research project
Development and exploration of this new and exciting direct RNA sequencing technique will provide tools with which to understand the effect that some mutations in breast cancer risk genes may have on patterns of gene expression.
Student: Jessie Chang
Supervisors:Dr Simone Cree, Dr Logan Walker, Allison Miller, Associate Professor John Pearson, Professor Martin Kennedy
Sponsor: TBC
Project brief
Introduction
Messenger RNA (mRNA) is a key component in the process of gene expression, which carries information from the genome to either guide the production of proteins, or to regulate genes through various molecular interactions.
The mRNA transcripts from most genes are assembled from discrete blocks, or exons, by a process called "splicing", which can result in several forms of transcript from each gene.
The different isoforms may produce proteins which differ in their function, and a subset of mutations can cause disease by altering these splicing patterns.
We are exploring a new technology called nanopore sequencing that enables direct analysis and sequencing of mRNA molecules, an approach that has only become commercially available in the past year. We are evaluating whether this method is effective for examining transcripts from important breast cancer risk genes such as BRCA1, BARD and BRIP1.
Aim
To explore and optimize methods for enriching the presence of RNA transcripts of interest, for targeted analysis by direct RNA sequencing on a nanopore sequencing device. This analysis will be targeted at genes that contribute to risk of breast cancer, such as BRCA1, BARD1 and BRIP, as well as control genes. Key goals will be:
- To test methods for enriching transcripts from selected genes, in a multiplexed approach
- To analyze the products of this enrichment strategy on either the MinION or GridION nanopore sequencer
- To examine the data with bioinformatic tools, and describe patterns of alternative splicing in these genes, and establish whether there is quantitative representation of transcripts from each gene
Method
This project will build on a prior BBiomedSc(Hons) project carried out in Prof Kennedy's lab. During that project, the student carried out initial direct RNA sequencing runs on the MinION, establishing that the method works effectively, enabling long reads of RNA transcripts derived from a cultured lymphoblastoid cell line. This summer project will further develop these methods, using RNA derived from cultured cell lines. RNA will be purified, then processed using kits from Oxford Nanopore.
Technologies (UK). Methods for enriching specific transcripts such as use of gene-specific primers, will be tested, and RNA samples will be run on either the MinION sequencer, or the GridION (a new, larger nanopore sequencer we are acquiring in mid-late 2018). Data will be captured, aligned and mapped with either minimap or Gmap, then visually examined for evidence of the enriched transcripts.
Student researcher's component of the study
The student will carry out all laboratory work and data analyses.
