MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes

Abstract

Characterization of dynamic DNA methylomes in diverse phylogenetic groups has attracted growing interest for a better understanding of the evolution of DNA methylation as well as its function and biological significance in eukaryotes. Sequencing-based methods are promising in fulfilling this task. However, none of the currently available methods offers the ‘perfect solution’, and they have limitations that prevent their application in the less studied phylogenetic groups. The recently discovered Mrr-like enzymes are appealing for new method development, owing to their ability to collect 32-bp methylated DNA fragments from the whole genome for high-throughput sequencing. Here, we have developed a simple and scalable DNA methylation profiling method (called MethylRAD) using Mrr-like enzymes. MethylRAD allows for de novo (reference-free) methylation analysis, extremely low DNA input (e.g. 1 ng) and adjustment of tag density, all of which are still unattainable for most widely used methylation profiling methods such as RRBS and MeDIP. We performed extensive analyses to validate the power and accuracy of our method in both model (plant Arabidopsis thaliana) and non-model (scallop Patinopecten yessoensis) species. We further demonstrated its great utility in identification of a gene (LPCAT1) that is potentially crucial for carotenoid accumulation in scallop adductor muscle. MethylRAD has several advantages over existing tools and fills a void in the current epigenomic toolkit by providing a universal tool that can be used for diverse research applications, e.g. from model to non-model species, from ordinary to precious samples and from small to large genomes, but at an affordable cost.

Correlation between HDAC4 enhancer DNA methylation and mRNA expression during palatal fusion induced by all‐trans retinoic acid

“…20 MethylRAD exhibits high specificity, sensitivity, reproducibility, and comparative economic cost and allows for de novo methylation analysis, extremely low DNA input, and flexible adjustment of tag density. 19 The current result shows the reliability of the MethylRAD-seq data of HDAC4 obtained from embryonic palates.…”

“…19,20 Clean reads were then subjected to pair-end sequencing on a HiSeq X Ten sequencer (Illumina Inc, San Diego, CA), according to the manufacturer's protocol, provided by the Shanghai Oebiotech Co Ltd (Shanghai, China). 19,20 Clean reads were then subjected to pair-end sequencing on a HiSeq X Ten sequencer (Illumina Inc, San Diego, CA), according to the manufacturer's protocol, provided by the Shanghai Oebiotech Co Ltd (Shanghai, China).…”

Opportunities and limitations of reduced representation bisulfite sequencing in plant ecological epigenomics

“…Furthermore, this approach is prohibitively expensive for large experimental designs, particularly for average to large sized plant genomes. Several recently developed approaches based on reducedrepresentation bisulfite sequencing methods (RRBS) provide a cost-effective alternative to WGBS, by interrogating only a representative fraction of the genome (Gu et al, 2011;Wang et al, 2015;van Gurp et al, 2016;Trucchi et al, 2016). RRBS approaches are scalable for ecological experimental designs, can be used on organisms without reference genomes, and allow greater resolution than previous marker-based approaches.…”

Climate Change Impacts on Seagrass Meadows and Macroalgal Forests: An Integrative Perspective on Acclimation and Adaptation Potential

“…While whole genome bisulfite sequencing is still expensive for large sample sizes, reduced representation techniques, such as RRBS (Gu et al, 2011), bsRADseq (Trucchi et al, 2016), and epiGBS (van Gurp et al, 2016) allow for cost-effective population epigenetic comparisons, partly without the need for a reference genome. Alternatively, DNA-methylation can be characterized with markers obtained via methylation-sensitive restriction enzymes, such as EpiRAD (Schield et al, 2016) and MethylRAD (Wang et al, 2015). While epigenetic population comparisons and stress responses can be studied for any species with DNAmethylation, an annotated reference genome is essential to infer the functional relevance of epigenetic differences and stress responses.…”