Protocol Matters: Which Methylome are You Actually Studying?

Abstract

The field of epigenetics is now capitalizing on the vast number of emerging technologies, largely based on second-generation sequencing, which interrogate DNA methylation status and histone modifications genome-wide. However, getting an exhaustive and unbiased view of a methylome at a reasonable cost is proving to be a significant challenge. In this article, we take a closer look at the impact of the DNA sequence and bias effects introduced to datasets by genome-wide DNA methylation technologies and where possible, explore the bioinformatics tools that deconvolve them. There remains much to be learned about the performance of genome-wide technologies, the data we mine from these assays and how it reflects the actual biology. While there are several methods to interrogate the DNA methylation status genome-wide, our opinion is that no single technique suitably covers the minimum criteria of high coverage and, high resolution at a reasonable cost. In fact, the fraction of the methylome that is studied currently depends entirely on the inherent biases of the protocol employed. There is promise for this to change, as the third generation of sequencing technologies is expected to again ‘revolutionize‘ the way that we study genomes and epigenomes.

KEYWORDS::

• DNA methylation
• epigenetics
• high-throughput sequencing
• tiling arrays

Acknowledgements

We thank Matthew Wakefield for helpful discussions of the technologies and data analysis, Dario Strbenac for technical assistance with the public sequencing datasets, Clare Stirzaker and Jenny Song for contributing the experiments using fully methylated DNA, and Rebecca Hinshelwood and Kate Patterson for the careful reading of the manuscript.

Financial & competing interests disclosure

This work is funded by National Health and Medical Res Council (NH & MRC) project (427614, 481347) (MDR, CS, DS) and Fellowship (SJC), Cancer Institute of New South Wales grants (CINSW: SJC; ALS), National Institute of Health grant 5R01 GM083084-03 (TPS) and National Breast Cancer Foundation Program Grant (SJC). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

This work is funded by National Health and Medical Res Council (NH & MRC) project (427614, 481347) (MDR, CS, DS) and Fellowship (SJC), Cancer Institute of New South Wales grants (CINSW: SJC; ALS), National Institute of Health grant 5R01 GM083084-03 (TPS) and National Breast Cancer Foundation Program Grant (SJC). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.