Nanopore sequencing of DNA from extracellular vesicles

Full Project Title

Developing a Nanopore diagnostic test using long DNA in extracellular vesicles for ovarian cancer chemoresistance, and complications in children with musculoskeletal disorders.

Project Summary

The development of methodologies utilising extracellular vesicle DNA (EV DNA) for diagnostic and prognostic purposes is a new area of research. In contrast, cell-free DNA is well characterised and is used routinely within pathology tests, such as non-invasive prenatal testing which uses placental cell-free DNA. Found in every bodily fluid examined, EVs contain cellular markers from difficult-to-access and viable anatomical sites, making them strong candidates as biomarkers of disease. For blood-based diagnostics, analysing DNA within EVs could resolve many of the known issues associated with use of cell-free DNA.

The aim of this project is to compare long EV DNA to short cell-free DNA using Nanopore long-read sequencing, and investigate whether the advantageous properties of EV DNA increases diagnostic accuracy through analyses such as genome phasing. This project will analyse mutation and methylation from long read sequencing data in specific biological contexts, with direct application to any field currently using EV DNA and circulating DNA biomarkers. To the best of our knowledge, no publications have used a long-read sequencing approach to analyse EV DNA, making this proposal highly novel.

In addition, we will leverage our initial data from long-read sequencing of EV DNA to develop a targeted sequencing method for use as a diagnostic test for clinical samples, representing a clear translational pipeline for this work.

Potential Outcomes

This project aims to develop a novel method to generate long-read mutation and methylation data from the ultra-low input DNA content within EVs. Project outcomes will provide guidance about the utility of long DNA within EVs, and whether this is advantageous to short cell-free DNA, in the context of molecular diagnostics. A pipeline for generating a targeted Nanopore sequencing method to enrich for DNA fragments linked to specific diagnoses will also be developed. If successful, these sequencing methods will have broad applicability to multiple fields in the clinical diagnostics space. In addition, these methods can be used as a tool by clinical researchers across UQ to further understand disease pathology caused by genomic abnormalities.