Parallel single-cell profiling now possible

New protocol allows links between DNA methylation and gene expression to be studied in the same cells

A new single-cell genomics method to study links between DNA modifications (methylation) and the activity of a gene has been developed. This is the first method that allows scientists to study the epigenome and transcriptome of a single cell in parallel, helping researchers pinpoint the relationship between changes in DNA methylation and gene expression.

The protocol, published in Nature Methods, is potentially transformative for epigenetics, as it reveals unprecedented detail of the epigenetic control of genes.

Single-cell sequencing technology has progressed rapidly in recent years, and is widely used to study how gene expression profiles (‘transcriptomes’) vary between cells. Recent single-cell protocols also allow researchers to explore chemical modification of DNA (‘epigenetics’), for example DNA methylation, which is a driving force behind changes to gene expression. Until now, it has only been possible to study single-cell transcriptomes and epigenomes separately.

“This new experimental protocol lets you assay both DNA methylation and RNA of the same single cell in parallel. Our approach provides the first direct view on the relationship between heterogeneity in DNA methylation and variation of expression in specific genes across single cells.”

Dr Oliver Stegle of the European Bioinformatics Institute (EMBL-EBI)

“This new method combines our previously developed protocol for parallel DNA and RNA sequencing with new advances in single-cell epigenetics. The result is an optimised approach that maximises the amount of biological information that can be obtained from a single cell.”

Dr Thierry Voet of the Wellcome Trust Sanger Institute and Katholieke Universiteit Leuven Belgium

To test the method, the group used mouse embryonic stem cells (ESCs) at a stage when they switch continuously between different gene-expression states. Just as in the cells of an early stage embryo, the identity of these cells is fluid rather than fixed. The researchers used two techniques in parallel: one that reveals detailed information about expression (how much variation there is, where that heterogeneity is coming from) and one to study DNA methylation in the same cells. For each cell, they obtained sufficient coverage to study epigenetic and transcriptome diversity of several thousand genes.

“The epigenetic state of ESCs is highly variable, and this variation is associated with changes in gene expression. Much of the transcriptional variability we see is thought to be driven by modifications of DNA, but now we have a technique that allows us to look at a single cell and discover relationships between DNA methylation and gene expression that were previously unknown. To understand development, it is really important that we pin these relationships down, and get them right.”

Professor Wolf Reik of the Sanger Institute and Babraham Institute

“Our statistical approach revealed hundreds of individual associations between variable epigenetic regions and gene expression. These associations can provide important insights into how pluripotency is maintained and how cell differentiation is regulated.”

Dr Christof Angermueller of EMBL-EBI

Going forward, the researchers expect the new protocol to offer new opportunities to study multiple different molecular layers simultaneously. This will go a long way towards understanding the connection between gene expression and DNA methylation in single cells, and identifying the factors that influence this relationship. Such research has implications for understanding normal development, and changes that occur with ageing and cancer.

The method was developed within the Sanger Institute/EMBL-EBI Single Cell Genomics Centre, a collaborative effort to develop single-cell technologies and apply them to new biological questions.

More information

Author contributions

Christof Angermueller, Stephen Clark, Heather Lee and Iain Macaulay are co-first authors on this work.

Funding

This research was funded by the BBSRC, the Wellcome Trust, the Medical Research Council, the European Union,
NoE EpiGeneSys and EMBL.

Participating centres

  • European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, Cambridge, UK
  • Epigenetics Programme, Babraham Institute, Cambridge, UK
  • Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK
  • MRC Functional Genomics Unit, University of Oxford, UK
  • Bioinformatics Group, Babraham Institute, Cambridge, UK
  • Department of Human Genetics, KU Leuven, Leuven, Belgium

Selected Websites

The Sanger/EMBL-EBI Single Cell Genomics Centre

The aim of the Sanger Institute/EMBL-EBI Single-Cell Genomics Centre is to develop and apply methods for capturing the complete genetic content of single cells in a high-throughput manner, enabling the exploration of cellular heterogeneity in normal development and disease. In addition to wet-lab approaches, we also develop the computational means for the analysis of single cells.

EMBL

EMBL is Europe’s flagship laboratory for the life sciences, with more than 80 independent groups covering the spectrum of molecular biology. EMBL is international, innovative and interdisciplinary – its 1800 employees, from many nations, operate across five sites: the main laboratory in Heidelberg, and outstations in Grenoble; Hamburg; Hinxton, near Cambridge (the European Bioinformatics Institute), and Monterotondo, near Rome. Founded in 1974, EMBL is an inter-governmental organisation funded by public research monies from its member states. The cornerstones of EMBL’s mission are: to perform basic research in molecular biology; to train scientists, students and visitors at all levels; to offer vital services to scientists in the member states; to develop new instruments and methods in the life sciences and actively engage in technology transfer activities, and to integrate European life science research. Around 200 students are enrolled in EMBL’s International PhD programme. Additionally, the Laboratory offers a platform for dialogue with the general public through various science communication activities such as lecture series, visitor programmes and the dissemination of scientific achievements.

EMBL-EBI

The European Bioinformatics Institute is part of EMBL, and is a global leader in the storage, analysis and dissemination of large biological datasets. EMBL-EBI helps scientists realise the potential of ‘big data’ by enhancing their ability to exploit complex information to make discoveries that benefit mankind. We are a non-profit, intergovernmental organisation funded by EMBL’s 21 member states and two associate member states. Our 570 staff hail from 57 countries, and we welcome a regular stream of visiting scientists throughout the year. We are located on the Wellcome Genome Campus in Hinxton, Cambridge in the United Kingdom.

The Babraham Institute

The Babraham Institute, which receives strategic funding (a total of £27.3M in 2014-15) from the Biotechnology and Biological Sciences Research Council (BBSRC), undertakes international quality life sciences research to generate new knowledge of biological mechanisms underpinning ageing, development and the maintenance of health. The Institute’s research provides greater understanding of the biological events that underlie the normal functions of cells and the implication of failure or abnormalities in these processes. Research focuses on signalling and genome regulation, particularly the interplay between the two and how epigenetic signals can influence important physiological adaptations during the lifespan of an organism. By determining how the body reacts to dietary and environmental stimuli and manages microbial and viral interactions, we aim to improve wellbeing and healthier ageing.

The Wellcome Trust Sanger Institute

The Wellcome Trust Sanger Institute is one of the world’s leading genome centres. Through its ability to conduct research at scale, it is able to engage in bold and long-term exploratory projects that are designed to influence and empower medical science globally. Institute research findings, generated through its own research programmes and through its leading role in international consortia, are being used to develop new diagnostics and treatments for human disease.

The Wellcome Trust

The Wellcome Trust is a global charitable foundation dedicated to achieving extraordinary improvements in human and animal health. We support the brightest minds in biomedical research and the medical humanities. Our breadth of support includes public engagement, education and the application of research to improve health. We are independent of both political and commercial interests.

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