Blood tubesFull title

Optimising Nanopore Sequencing for Genome-wide DNA Methylation Analysis in Cell-free DNA

Aim

The aim of this project is to develop a comprehensive and reproducible protocol for analysing genome-wide DNA methylation in cell-free DNA (cfDNA) using Oxford Nanopore sequencing. Traditional methods of genome-wide DNA methylation analysis through sequencing involve bisulfite conversion, but with levels of cfDNA already so low this is not always feasible. Therefore, the goal of this project is to optimise a cfDNA sequencing protocol that does not require bisulfite conversion, and that can be conducted in real-time for future diagnostic applications.

Brief Project Outline

cfDNA is being widely investigated as a diagnostic biomarker for a range of clinical conditions including cancer and cardiovascular disease. DNA methylation analysis is crucial for identifying the source of circulating cfDNA fragments, enabling the use of cfDNA as a biomarker for organ-specific injury. However, there is currently no published research exploring methylation analysis in cfDNA via Nanopore sequencing. As such, this project will optimise the use of Nanopore technology for cfDNA sequencing.

The following aims need to be addressed in order to optimise Nanopore sequencing for methylation analysis in cfDNA:

  1. Identifying optimal quantity of input cfDNA;
  2. Assessing of the feasibility of multiplexing samples;
  3. Identifying appropriate modifications of existing Nanopore library preparation kits for efficient cfDNA sequencing; and
  4. Comparison of genome aligners and methylation analysis pipelines for short read alignment and subsequent DNA methylation calling.

This project will utilise samples collected as part of a wider study looking at cfDNA as a biomarker for cardiovascular disease. The Nanopore sequencing output will be validated against Whole Genome Bisulfite Sequencing (WGBS) which is the current gold standard for genome-wide methylation analysis. Nanopore sequencing presents an exciting opportunity for low cost, real-time, and efficient sequencing of cfDNA, but requires thorough optimisation before application.

Genomics-based innovative aspect of proposal

Traditional methods of genome-wide DNA methylation analysis through sequencing involve bisulfite conversion (the current gold standard), and with levels of cfDNA already so low, this methods is not always feasible. The ability to analyse DNA methylation in cfDNA, both without the need for bisulfite conversion and in real time, has incredible potential for clinical translation. This project aims to utilize current GIH expertise in Nanopore sequencing, while also extending GIH’s capabilities to include optimising Nanopore sequencing and DNA methylation analysis for cfDNA and other short fragments.

Broad applicability of the technique

The ability to analyse methylation in cfDNA plays a crucial role in identifying the origin of the circulating DNA fragments and specific abnormalities that may be associated with cancer or disease progression. Therefore, there is significant potential for this technique to be applied to a variety of different conditions including cancer, organ failure, and other injury-related diseases, not only to monitor progression but also for early-diagnosis and potentially even to inform prognosis. There are several groups in UQ who are conducting research on cfDNA for whom this technique may be directly applicable. In addition, we are aiming to publish the methodology so that it available not only for these groups, but for the international research community as a whole.

Project members

Research collaborators

Janice Reid

Janice Reid

PhD candidate
Critical Care Research Group (CCRG), Faculty of Medicine
Dr Devika Ganesamoorthy

Dr Devika Ganesamoorthy

Research Fellow
Child Health Research Centre, Faculty of Medicine

Kieran Hyslop

Research Assistant
Critical Care Research Group (CCRG), Faculty of Medicine
Professor John Fraser

Professor John Fraser

Professor
Prince Charles Hospital Northside Clinical Unit, Faculty of Medicine
Dr Jacky Suen

Dr Jacky Suen

Adv Qld Industry Research Fellow & Senior Research Fellow
Prince Charles Hospital Northside Clinical Unit, Faculty of Medicine
Affiliate Senior Research Fellow
School of Biomedical Sciences, Faculty of Medicine

Genome Innovation Hub

Valentine Murigneux

Valentine Murigneux

Senior research assistant
Genome Innovation Hub
Bioinformatician
QCIF Facility for Advanced Bioinformatics
Dr Subash Rai

Dr Subash Rai

Research Assistant
Genome Innovation Hub