Background

With the development of high-throughput sequencing technologies, evaluation of genome-wide DNA methylation profiles is more accessible than before. For the human genome, various commercial targeted methyl capture sequencing panels and methylation arrays are available and allow high-throughput, quantitative interrogation of methylation sites at single-nucleotide resolution. However, most of these commercial tools are not offered for other model species. Thus, cost-effective sequencing-based methods for genome-wide methylation analysis, not targeting species-specific genomic sequences, are still highly relevant and valuable especially when profiling large cohorts of samples. One of these techniques is Reduced Representation Bisulfite Sequencing (RRBS). This method enables you to quantitatively investigate the DNA methylation profile of ~10% of the overall CpGs, clustered in fragments composing approximately 1% of the genome, with a preference for CpG rich regions (e.g., CpG islands, promoters) (Meissner et al., 2005 and 2008; Smith et al., 2009; Gu et al., 2011).

Here, we present a rapid RRBS (rRRBS) protocol (Piché et al., 2018) that significantly decreases hands-on time, allowing to perform the entire protocol in ~2 days compared to the 7 days usually needed (Gu et al., 2011; Boyle et al., 2012). Importantly, by using a qPCR amplification step, we now minimize amplification bias during multiplexed library construction (Figure 1). Also, compared to the original and other RRBS protocols (Gu et al., 2011; Boyle et al., 2012; McGraw et al., 2015) our modified protocol reduces the quantity of reagent and enzyme used as we no longer test for the optimal number of cycles needed before the final library amplification step. This method is suitable for DNA methylation studies from cells or tissues from any common research species (e.g., human, mouse, rat, zebrafish).


Figure 1. Graphical summary of the principal step of the rRRBS procedure