Methylation content sensitive enzyme ddRAD (MCSeEd): a reference-free, whole genome profiling system to address cytosine/adenine methylation changes

Gianpiero Marconi,Alberto Acquadro,Emidio Albertini,Cinzia Comino,Stefano Capomaccio,Ezio Portis,Andrea Porceddu

doi:10.1038/s41598-019-51423-2

Gianpiero Marconi, Alberto Acquadro + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-51423-2

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Abstract

Methods for investigating DNA methylation nowadays either require a reference genome and high coverage, or investigate only CG methylation. Moreover, no large-scale analysis can be performed for N6-methyladenosine (6 mA) at an affordable price. Here we describe the methylation content sensitive enzyme double-digest restriction-site-associated DNA (ddRAD) technique (MCSeEd), a reduced-representation, reference-free, cost-effective approach for characterizing whole genome methylation patterns across different methylation contexts (e.g., CG, CHG, CHH, 6 mA). MCSeEd can also detect genetic variations among hundreds of samples. MCSeEd is based on parallel restrictions carried out by combinations of methylation insensitive and sensitive endonucleases, followed by next-generation sequencing. Moreover, we present a robust bioinformatic pipeline (available at https://bitbucket.org/capemaster/mcseed/src/master/) for differential methylation analysis combined with single nucleotide polymorphism calling without or with a reference genome.

Highlights

The MCSeEd technique was used to monitor DNA methylation changes induced by drought stress in maize leaves
We have developed a reduced-representation, reference-free approach for characterizing whole genome methylation patterns across different methylation contexts
Alternative approaches aimed at reducing the sequencing demands are based on genomic digestion with either methylation-sensitive or methylation-insensitive endonucleases, before direct next-generation sequencing (NGS) (e.g., EpiRADseq[30], MRE-seq29), or bisulfite treatments and NGS (e.g., RRBS27, methylation-sensitive restriction enzyme bisulfite sequencing [MREBS]41)

Summary

Objectives

Since one of our goals was to develop a high-throughput technique that can be applied to species without a reference genome, the MCSeEd bioinformatic pipeline was tested for mapping of filtered reads to a pseudo-reference genome autogenerated by a pipeline, hereafter indicated as the genome independent strategy

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