Recent developments in DNA methylation analysis technologies have made it crucial for researchers to understand which tool is optimal for their epigenetic research. These new methods pose exciting opportunities never before imagined, allowing for epigenetic variation to be connected to phenotypic consequences on a much grander scale and at single-base resolution. In a recent issue of an epigenetics newsletter, The Decoder, scientists at Epigentek discuss the latest progress made in profiling genome-wide and region-specific DNA methylation and offer suggestions on choosing the best method specifically for your experiment.
They begin by describing a method called oxidative bisulfite-sequencing (oxBS-Seq), which now enables researchers to distinguish 5-hydroxymethylcytosine (5-hmc) from 5-methylcytosine (5-mc). It takes advantage of the chemical oxidation (KRuO4) of 5-hmc into 5-fC and ensures only 5-mc is detected by converting 5-fC to uracil by bisulfite treatment. Traditional bisulfite sequencing, alternatively, results in incorrectly identifying DNA methylation status, since both 5-mC and 5-hmC are bisulfite converted identically (C to U). The oxidation step allows 5-hmC bases to be subtracted out.
The recent development of post-bisulfite sequencing overcomes limitations of whole-genome bisulfite sequencing (WGBS). According to the experts at Epigentek, it is particularly helpful to researchers who are unable to use large amounts of DNA (>1 µg) which may be hard to come by when testing, for instance, tumor biopsy samples, early embryos, or circulating DNA. Post-bisulfite sequencing ensures maximum yields from starting materials as little as a single cell. In this method, DNA is treated with bisulfite salt and then converted to double-stranded DNA for ligation and PCR amplification.
Targeted methylation-sequencing solves the issue of limited flexibility and efficiency of DNA methylation assays for characterizing multiple genomic targets. To carry out this technique, you can use different methods which vary in specificity and sensitivity: bisulfite-PCR amplification, ligation capture, bisulfite padlock probe (BSPP) capture or liquid hybridization followed by bisulfite-sequencing. Bisulfite-PCR amplification requires primers that are based on the bisulfite converted reference genome. A maximum of 20,000 loci can be targeted by performing multiple singleplex PCR amplifications in emulsion droplets. Another option for targeted methylation-sequencing, ligation capture, is based on reducing the complexity of the genomic DNA for analysis by selected enrichment of target genome regions. BSPP capture uses padlock probe-based capture of bisulfite-converted DNA and, as a unique feature, probes can be pooled into a single capturing reaction. Liquid hybridization based capture for bisulfite-sequencing (LHC-BS) entails hybridization of preligated target DNA fragments into biotinylated oligonucleotides followed by affinity enrichment selection, bisulfite conversion, PCR amplification and sequencing. Using this method, compared to ligation capture and BSPP capture, ensures more uniform target coverage.
Epigenetic editing, a type of genetic engineering, has grown in popularity since it was recently developed. It allows for precise, targeted editing in which the epigenome can be modified at specific sites. This groundbreaking development enables researchers to determine the exact biological role of an epigenetic modification – such a DNA methylation or histone methylation – at a particular locus of interest. The CRISPR/Cas9 system is used for epigenetic editing and is successful in areas in which earlier methods, such as a Zinc finger system or TALE system, are not. In this system, histone or DNA modifying enzymes are joined to catalytically inactive Cas9 (dCas9), which can still be recruited by gRNAs without cleaving the DNA. This allows for the modulation of epigenetic states of specific DNA sites.
Selecting the right tool for your epigenetic research can be difficult in light of these new sequencing-based advances and an ever-growing repertoire of DNA methylation analysis methods. Although cost, minimum sample input requirements, accuracy, rapidity and throughput remain important considerations, the key to a truly successful analysis lies in selecting the proper tool for the job.
To learn which tool would be most optimal for use in your epigenetic research, read the entire Decoder article (Issue 3) here: http://www.epigentek.com/catalog/newsletter.php