A team of researchers from the University of Toronto, the Ontario Institute for Cancer Research and Johns Hopkins University have developed software to detect cytosine methylation in DNA through nanopore sequencing. The scientists used MinION, a compact, pen drive-shaped sequencer developed by Oxford Nanopore Technologies to analyze the methylome with no need for specific steps for library preparation, according to the study published in Nature Methods. The technique will help directly characterize the epigenetic modifications in DNA from small tissue samples.
“Nanopore sequencing allows methylation data to come along with sequencing ‘for free’ - meaning that we get this data along with any mutation or structural variation data. This adds richness to the data,” says Jared T. Simpson, lead author of the article and researcher at the Ontario Institute for Cancer Researc, in remarks to Biocores. “Secondly, it gives us an idea of long-range, single-molecule methylation patterns, giving clues to how methylation is regulated,” he adds. His technique also offers other advantages over conventional methods.
Currently, when mapping cytosine methylation in DNA, scientists tend to use bisulfite sequencing, a procedure that enables recognition of the allele-specific methylations on CpG islands. This treatment deaminates the DNA’s unmethylated cytosines, turning them into uracil, but does not affect the methylated cytosines (or 5-methylcytosines) because of their physical-chemical properties. Though considered the gold standard for mapping, the technique can cause degradation of the DNA and other analysis problems, forcing researchers to work with large amounts of DNA (over 100 ng, approximately).
Source: Johns Hopkins University
“The advantages of our method over bisulfite sequencing is a direct detection—with no additional chemical or enzymatic treatment—of modified nucleotides,” states Simpson. The software developed enables researchers to discern with 95% accuracy the methylated from the unmethylated cytosines in a single step, although at present it does not distinguish other types of modifications, according to the Nature Methods article. The University of Toronto, Ontario Institute for Cancer Researc and Johns Hopkins University researchers tested their technique in mapping the methylome of several cell lines and checked their results against those registered using bisulfite sequencing. “Nanopore sequencing has long reads, allowing for phased methylation—patterns of methylation over large, multi-kb stretches of the genome—even allele-specific methylation,” states Jared T. Simpson in remarks to Biocores.
This is not the first time that nanopore sequencing has been used to determine cytosine methylations, an application already performed by teams at the University of Washington and the University of California Santa Cruz, both of which published articles, in PNAS and BioRxiv respectively. “But we have developed software to make it practical to apply methylation classification to biological samples using the commercial nanopore sequencing (MinION, Oxford Nanopore Technologies),” clarifies Simpson. Simpson’s group was capable of analyzing different DNA samples from breast cancer cell lines, and successfully detected the epigenetic modifications that had taken place.
The main advantage that nanopore sequencing provides in identifying the cytosine methylations is that it is an easy method that is comfortable to use, and not only offers information on epigenetic marks in DNA but also performs a complete reading of the genome at the same time. Nevertheless, according to Simpson, the technique is still a little slower than standard nanopore sequencing. Specifically, it is more costly than Ilumina technology per base pair analyzed. Further, it does not recognize other modifications in the epigenome such as 5-hmC, 5-formylcytosine (5-fC), 5-carboxylcytosine (5-caC) or damages to DNA (8-oxoguanine). The software, available for free at GitHub for anyone wishing to use it, has a number of mid and long-term applications. Among other uses, the authors point out that epigenetic mapping will facilitate improved characterization of cancer, the identification of methylations in plants and the analysis of the bacterial methylome.