Exploring genome wide bisulfite sequencing for DNA methylation analysis in livestock: a technical assessment.

Rachael Doherty,Christine Couldrey

doi:10.3389/fgene.2014.00126

Abstract

Recent advances made in “omics” technologies are contributing to a revolution in livestock selection and breeding practices. Epigenetic mechanisms, including DNA methylation are important determinants for the control of gene expression in mammals. DNA methylation research will help our understanding of how environmental factors contribute to phenotypic variation of complex production and health traits. High-throughput sequencing is a vital tool for the comprehensive analysis of DNA methylation, and bisulfite-based strategies coupled with DNA sequencing allows for quantitative, site-specific methylation analysis at the genome level or genome wide. Reduced representation bisulfite sequencing (RRBS) and more recently whole genome bisulfite sequencing (WGBS) have proven to be effective techniques for studying DNA methylation in both humans and mice. Here we report the development of RRBS and WGBS for use in sheep, the first application of this technology in livestock species. Important technical issues associated with these methodologies including fragment size selection and sequence depth are examined and discussed.

Highlights

DNA methylation analysis has become an important component of the post-genomic agricultural research era
In human and mouse research, the application of reduced representation bisulfite sequencing (RRBS) methods have allowed for genome wide DNA methylation analysis with reduced sequencing requirements, thereby making studies with multiple replicates, group comparisons or cohort studies more achievable and affordable (Boyle et al, 2012)
QUALITY CONTROL AND MAPPING EFFICIENCIES FOR LIBRARIES PREPARED FROM DIFFERENT DNA FRAGMENT LENGTHS Quality control analysis using FastQC indicated that for all three fragment sizes analyzed by RRBS, the 100 bp sequences displayed the expected nucleotide composition

Summary

Introduction

DNA methylation analysis has become an important component of the post-genomic agricultural research era. The RRBS methodology, designed by Meissner et al (2005), Gu et al (2011) allows for preferential selection and sequencing of CpG-rich regions whilst CpG-poor intergenic regions are under-represented in the library. This results in the sequencing of a subset of DNA fragments from the genome which is likely to contain the majority of regions relevant for DNA methylation analysis without the sequencing of regions that are devoid of CpG sites reducing the cost. By using or combining different restriction enzymes, CpG coverage across the genome can be altered to include or exclude certain regions of interest such as CpG island shores, which are known to play an important role in various biological processes including cellular differentiation (Doi et al, 2009; Wang et al, 2013)

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