Long-read and single-cell DNA epigenetic sequencing to study cancer heterogeneity

Project Overview

Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution

Background

Single-cell RNA sequencing has become a powerful tool to study cellular heterogeneity. However, the transcriptome is easily influenced by the environment and does not reveal the underlying mechanisms of gene regulation. DNA methylation is one of the best-studied epigenetic information in mammals regulating gene expression. It is dynamically regulated in development, cell type, and tissue-specific, and yet relatively stable, which makes it ideally positioned to study cellular plasticity and heterogeneity, especially at long-read and single-cell level. However, current long-read DNA methylation sequencing is not accurate and single-cell DNA methylation sequencing heavily relied on bisulfite sequencing, which uses harsh chemical conditions intrinsically unfriendly to low-input samples, resulting in low quality and sparse data.

Aims and Objectives

Recently, we developed TAPS (TET-Assisted Pyridine borane Sequencing), a bisulfite-free and direct DNA methylation and hydroxymethylation sequencing method, which employed mild reactions that is non-destructive and can perverse long DNA fragments. Building on this revolutionary method, this project aims to develop long-read TAPS and sing-cell TAPS delivering high quality and rich data to study cellular heterogeneity. TAPS also provides an excellent starting point to develop multi-omics integration of genome, transcriptome, and epigenome to better dissect the gene regulation mechanisms. These tools will be applied to characterize the heterogeneity of cells in oesophageal cancer samples in response to immunotherapy with the goal of identifying epigenetic targets for the development of novel diagnostics and therapeutics.

Training Opportunities

In addition to a wide range of basic and advanced biochemistry, cell and molecular biology techniques, the project offers training in cutting-edge technologies, including second and third-generation sequencing, sing-cell techniques, and bioinformatics data analysis.

Theme

Genetics & Genomics and Cancer Biology

Admissions

Project reference number: 985

Funding and admissions information

Supervisors

Name Department Institution Country Email
Chunxiao Song Oxford Ludwig Institute Oxford University, NDM Research Building GBR chunxiao.song@ludwig.ox.ac.uk
Xin Lu Oxford Ludwig Institute Oxford University, Old Road Campus Research Building GBR xin.lu@ludwig.ox.ac.uk
Skirmantas Kriaucionis Oxford Ludwig Institute Oxford University, Old Road Campus Research Building GBR skirmantas.kriaucionis@ludwig.ox.ac.uk

Liu Y, Siejka-Zielińska P, Velikova G, Bi Y, Yuan F, Tomkova M, Bai C, Chen L, Schuster-Böckler B, Song CX. 2019. Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution. Nat. Biotechnol., 37 (4), pp. 424-429. Read abstract | Read more

Bisulfite sequencing has been the gold standard for mapping DNA modifications including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) for decades. However, this harsh chemical treatment degrades the majority of the DNA and generates sequencing libraries with low complexity. Here, we present a bisulfite-free and base-level-resolution sequencing method, TET-assisted pyridine borane sequencing (TAPS), for detection of 5mC and 5hmC. TAPS combines ten-eleven translocation (TET) oxidation of 5mC and 5hmC to 5-carboxylcytosine (5caC) with pyridine borane reduction of 5caC to dihydrouracil (DHU). Subsequent PCR converts DHU to thymine, enabling a C-to-T transition of 5mC and 5hmC. TAPS detects modifications directly with high sensitivity and specificity, without affecting unmodified cytosines. This method is nondestructive, preserving DNA fragments over 10 kilobases long. We applied TAPS to the whole-genome mapping of 5mC and 5hmC in mouse embryonic stem cells and show that, compared with bisulfite sequencing, TAPS results in higher mapping rates, more even coverage and lower sequencing costs, thus enabling higher quality, more comprehensive and cheaper methylome analyses. Hide abstract