A cost-effective approach to DNA methylation detection by Methyl Sensitive DArT sequencing.

Wendell Jacinto Pereira,Dario Grattapaglia,Marília De Castro Rodrigues Pappas,Georgios Joannis Pappas,Andrei Chernov

doi:10.1371/journal.pone.0233800

Wendell Jacinto Pereira, Dario Grattapaglia + Show 3 more

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https://doi.org/10.1371/journal.pone.0233800

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Abstract

Several studies suggest the relation of DNA methylation to diseases in humans and important phenotypes in plants drawing attention to this epigenetic mark as an important source of variability. In the last decades, several methodologies were developed to assess the methylation state of a genome. However, there is still a lack of affordable and precise methods for genome wide analysis in large sample size studies. Methyl sensitive double digestion MS-DArT sequencing method emerges as a promising alternative for methylation profiling. We developed a computational pipeline for the identification of DNA methylation using MS-DArT-seq data and carried out a pilot study using the Eucalyptus grandis tree sequenced for the species reference genome. Using a statistic framework as in differential expression analysis, 72,515 genomic sites were investigated and 5,846 methylated sites identified, several tissue specific, distributed along the species 11 chromosomes. We highlight a bias towards identification of DNA methylation in genic regions and the identification of 2,783 genes and 842 transposons containing methylated sites. Comparison with WGBS, DNA sequencing after treatment with bisulfite, data demonstrated a precision rate higher than 95% for our approach. The availability of a reference genome is useful for determining the genomic context of methylated sites but not imperative, making this approach suitable for any species. Our approach provides a cost effective, broad and reliable examination of DNA methylation profile on MspI/HpaII restriction sites, is fully reproducible and the source code is available on GitHub (https://github.com/wendelljpereira/ms-dart-seq).

Highlights

In the context of a common DNA sequence, cells employ different strategies to coordinate the set of expressed and repressed genes in order to establish cell identity and fate during development and physiological state, as well as adaptation to external stimuli
The featured MS-DArT-seq methodology is a 5mC profiling method that can be classified under the umbrella of genome reduction by means of methylation-sensitive restriction enzyme digestion, such as methyl-sensitive cut counting (MSCC) [25] or methyl sensitive restriction enzymes (MREs)-seq [29]
Our approach branches out from these established techniques as it relies on double digestion with the use of the RE PstI and one of each of the isoschizomers MspI and HpaII that recognize the prototypical site (CCGG) covering CG and CHG methylation contexts

Summary

Introduction

In the context of a common DNA sequence, cells employ different strategies to coordinate the set of expressed and repressed genes in order to establish cell identity and fate during development and physiological state, as well as adaptation to external stimuli. A cost-effective approach to DNA methylation detection by MS-DarT-seq. Embrapa Genetic Resources and Biotechnology provided support in the form of salaries for authors MRP and DG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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