Epigenetics, the study of heritable phenotypic changes that are not attributable to changes in DNA sequence, is a rapidly advancing field that is increasingly providing insight into a range of genetic phenomena. Epigenetic changes regulate gene expression independent of genetic sequence. Questions addressed by the field include cell differentiation, tumorogenesis and expression of traits inherited maternally versus paternally. Recent research publications demonstrate the diversity of applications and research areas the discipline covers as well as the range of technologies used for its analyses.

Several types of epigenetic processes occur, including DNA base modification, with methylation, the addition of a methyl group to a cytosine base, the most studied. Methylation is involved in the regulation of gene expression and has been implicated in processes such as cancer development and protecting bacterial cells from foreign DNA. Sequencing is an important technology for methylation analysis, and Illumina and Pacific Biosciences, two companies whose platforms are used for epigenetic sequencing, each has its own approach to this shared application.

The most common type of methylation forms 5-methylcytosine (5mc), frequently found at a cytosine followed by a guanine (CpG). CpG islands, or areas with a high concentration of CpG sites, often occur at promoter regions, where DNA transcription is initiated. Methylation at these regions usually silences gene expression. Another base modification beginning to receive much research attention is 5-hydroxymethylcytosine (5hmc), an oxidized form of 5mc. It is found at high levels in brain and stem cells and, among other potential functions, can play a role in demethylation.

Illumina’s epigenetic-sequencing approach supports bisulfite-replacement sequencing, which enables methylation to be studied at single-base-pair resolution. DNA is treated with sodium bisulfite, which converts cytosine to uracil. However, methylated cytosines are protected from conversion. Sequencing the resulting DNA reveals which bases were methylated. Bisulfite sequencing can be used to study methylation over the whole genome (whole-genome bisulfite sequencing, or WGBS) or in a targeted fashion (reduced-representation bisulfite sequencing, or RRBS). Complications of bisulfite replacement may include the loss of much of the sample DNA due to treatment with harsh chemicals and the inability to distinguish between 5mc and 5hmc.

John Greally, MD, PhD, director, Center for Epigenomics, Albert Einstein College of Medicine, researches epigenetic regulation in the human genome and its applications to human disorders such as autism and extreme fetal growth. He told IBO, “I’m very much in favor of doing [bisulfite] sequencing-based approaches because they offer not just good quantitative resolution but also a lot of qualitative information that you really can’t get from microarrays [which are also frequently used for methylation research].” For example, he explained, sequencing can be used to study chromatin structure and small and noncoding RNAs, both of which are involved in other epigenetic processes.

Dr. Greally said that survey approaches, including both RRBS and microarray analysis, have focused on methylation of promoter sequences. However, with respect to the role of methylation in phenomena such as cell differentiation and disease, “[W]e really need to look at the more distal enhancers that are in the genome.” According to Dr. Greally, this is most effectively performed with WGBS.

Illumina’s bisulfite-sequencing approach to epigenetics is widely used for methylation analysis on eukaryotes. Study systems in the publications listed on Illumina’s website include human, mouse and rice, and study areas include characterization of tumor development, cell development and disease, age-related cellular changes and inheritance of environmental information. The company provides products for both WGBS and RRBS as well as microarrays for methylation studies. Its TruSeq DNA Methylation Kit, which denatures genomic DNA to single stranded after treatment with sodium bisulfite, is used to prepare libraries for WGBS.

Although, as noted above, bisulfite sequencing cannot distinguish between 5mc and 5hmc, Britt Flaherty, PhD, associate product manager, Epigenetics, at Illumina indicated customers may still do so via treatments prior to bisulfite sequencing. “They do this through alternative chemistries like TAB-seq [Tet-assisted bisulfite sequencing] and OxBS [oxidative bisulfite sequencing].” She continued, “Some labs perform these additional experiments on a large scale across the whole genome through Illumina sequencing, while others just sequence the region of interest for confirmation.” Regarding degradation of sample DNA as a result of bisulfite treatment, Dr. Flaherty stated that Illumina’s customers have successfully used “standardized samples [to] control for and identify experimental variance.”

Dr. Greally commented that although the methods of identifying 5hmc via bisulfite sequencing work well, the proportion of sequencing reads with 5hmc tend to be very low. “So even 50x coverage . . . that we’d normally be happy with for DNA methylation studies appears not to offer the dynamic range that you would need to be able to distinguish hydroxymethylation coming from two cell states [such as two types of cells or normal versus diseased cells],” he said. “Whole-genome approaches for hydroxymethylation are going to be very, very difficult because you need enormous coverage,” he continued. “So what we’re all in the field looking at is how to target deep sequencing to loci that are probably going to be informative for hydroxymethylation.”

The growth of methylation analysis and Illumina’s status in the market are reflected in the demand for the company’s epigenetic-sequencing products. “We are seeing an uptick in large-scale studies in sequencing across all fields. . . . The [NIH] Roadmap Epigenomics [Mapping Consortium] is one such example,” said Dr. Flaherty. The consortium promotes basic and disease-related research by compiling epigenomic data from human tissues and cells. “Researchers profiled hundreds of isolated cell types through a combination of bisulfite sequencing and ChIP [chromatin immunoprecipitation, used to study DNA-protein interactions by enriching for protein-bound DNA] assays to establish a public resource,” she stated. “This is no longer a targeted field—every genetic study touches on epigenetics in some way.”

Illumina’s outlook on epigenetics extends beyond methylation studies. According to Dr. Flaherty, “[M]ethylation arrays and sequencing will both continue to drive this field. Many customers are looking at epigenetics through ChIP-seq [sequencing of DNA enriched via ChIP], though, to understand protein-DNA interaction. Robust immunoprecipitation assays with strong antibodies will be critical to the research in this field.“

With its single-molecule-real-time (SMRT) sequencing, Pacific Biosciences fills a different role in epigenetic-sequencing research. The company’s technology allows DNA base modifications to be detected based on polymerase kinetics during sequencing. During DNA synthesis, the sequencing polymerase slows down if a base in the template is modified. The level of slowdown varies by the type of modification. Based on these kinetics, SMRT sequencing can identify more than 25 modifications, including distinguishing between 5mc and 5hmc.

Pacific Biosciences’ market position in methylation sequencing is in studies of small genomes, mostly bacteria, as a result of the technology’s lower throughput. According to Jonas Korlach, PhD, CSO, Pacific Biosciences, studies of bacterial pathogenicity are a common application of SMRT epigenetic sequencing. For example, based on methylation patterns, the technology allowed researchers to discover that the increased virulence of a particular strain of E. coli was likely due in part to its incorporation of a methylase, which adds methyl groups to molecules, from a bacteriophage, thus changing the gene regulation of the bacterium.

The company is also establishing its epigenetic-sequencing offerings beyond the scope of research applications, such as in applied and industrial microbiology. “Understanding the methylation pattern allows you not only to understand better certain . . . biotechnologically useful activities of bacteria but also allows you to better manipulate them,” said Dr. Korlach. For example, this understanding, he explained, is important in the dairy industry to gain insight into issues including why probiotics are beneficial, how bacteriophages can ruin bacterial cultures in dairy products and in the development of new products. Dr. Korlach also named bioremediation and the development of natural products, such as optically pure lactic acid, as up-and-coming applications of SMRT epigenetic sequencing. He speculated that the technology might also help identify pathways unique to bacteria, which could be targeted to develop new antibiotics.

The use of SMRT sequencing for epigenetic research has also been expanded to some extent to eukaryotic systems. For example, researchers have used SMRT sequencing to study methylation in mitochondrial genomes, and the technology has also been applied to mouse stem cells. “I would certainly anticipate that that’s going to become much more common,” Dr. Korlach said. “This is something that’s now being revisited [since the company’s latest chemistry and software release (see IBO 10/31/14)] because the throughput is now much better.” He added that he knows several researchers studying plant and animal genomes who are beginning epigenomics studies using Pacific Biosystems’ platform. Dr. Korlach predicts that it will become standard for scientists to study epigenomes along with genomes. “Hopefully, [they] will look at the types of biological questions more in a holistic context of not really seeing them as being separate, but being . . . part of one scientific study,” he said.

Dr. Greally also shed light on potential expanded uses of SMRT epigenetic sequencing. For example, he said, outside of CpG islands, where 99% of methylation occurs, the frequency of CpGs is only about one in a hundred base pairs. Short-read sequencing, such as Illumina’s, will thus detect perhaps two of those CpGs on average. Researchers are beginning to realize that the relationship of methylation across CpG sites on the same strand of DNA may provide information on phenomena such as cancer, he explained. If SMRT sequencing is able to walk through 10 kb of the genome and detect tens to hundreds of CpGs, for example, the technology will prove valuable, according to Dr. Greally.

Dr. Greally predicts that the field of epigenomics as a whole will become all sequencing based. “[Researchers] are going to be doing probably things like whole-genome sequencing using [Illlumina’s HiSeq] X Ten machine; they’ll be doing DNA methylation studies; they might do ATAC-seq (assay for transposase-accessible chromatin with sequencing); they’ll do transcriptional profiles as well,” he said. “[I]t won’t be just looking at DNA methylation anymore.”