Tweet
Developments in sequencing technologies and methodologies have transformed the field of epigenetics, giving researchers a better way to understand the complex world of gene regulation and heritable modifications. This article explores some of the diverse sequencing methods employed in the study of epigenetics, ranging from classic techniques to cutting-edge innovations while providing a brief overview of their processes, applications, and advances.
Methylation Detection
DNA methylation is an important regulator of gene expression, which is often studied by the identification and quantification of methylated cytosines. When it comes to studying complex patterns of methylation, “Methyl-Seq is the most traditional approach,” said Andrea O’Hara, Ph.D., Strategic Technical Specialist at Azenta Life Sciences. “Depending on the cell type or sample type, there may be extensive previous knowledge in this area to build off of; however, it is typically done at the whole genome level, which yields tremendous amounts of data.”
Methyl-Seq is extensively used in a wide range of applications because it “enables comprehensive analysis of genome-wide methylation at single-base resolution,” added O’Hara. “It can be customized to target specific regions of interest and/or specific types of methylation residues.”
This method requires a multi-step process that involves enzymatically or chemically converting unmethylated cytosines to uracils. These base changes are detected during sequencing by comparing treated and untreated samples, revealing the original methylation patterns. Any specific changes in methylation states can then be measured by comparing two samples to determine important differences.
Scientists continue to regularly use Methyl-Seq for their research, but popular alternatives to this method include utilizing long-read sequencing technologies such as Oxford Nanopore Technologies and Pacific Biosciences instruments that can directly detect methylation. These platforms are now able to distinguish modified bases from their unmodified counterparts during the sequencing process, and when combined with their long reads can provide information such as allele-specific methylation, long-range interactions, and other important DNA methylation patterns.
Chromatin Accessibility
Chromatin accessibility refers to the availability of DNA regions for regulatory proteins, providing insights into the potential for gene regulation and the activity of specific genomic elements. The method most commonly used to determine chromatin accessibility is ATAC-Seq (assay for transposase-accessible chromatin with high-throughput sequencing).
ATAC-Seq employs a Tn5 transposase that fragments DNA at open chromatin regions while simultaneously adding sequencing adapters. These fragments are sequenced and the open regions are identified during data analysis. ATAC-Seq is valuable because it “sequences the open areas of chromatin that are accessible for transcription,” explained O’Hara. “When coupled with RNA-Seq to measure RNA expression, one can easily interpret which accessible areas are actively transcribed within any particular sample.”
Compared to alternative methods like MNase-Seq, FAIRE-Seq, and DNase-Seq, ATAC-Seq is often preferred due to its improved sensitivity. Furthermore, researchers often favor ATAC-Seq because it “employs Tn5 transposase to identify open chromatin with a higher signal-to-noise ratio, requires no background knowledge of the chromatin states for analysis, and can be processed in bulk or single-cell analyses,” explained O’Hara.
The combination of ATAC-Seq with single-cell sequencing is one of the biggest advances in the method. “Single-cell ATAC-Seq is a particularly powerful application,” stated O’Hara. With currently available multiomics kits, “one can measure the ATAC-Seq and RNA-Seq profile from thousands of individual cells in a sample. This allows researchers to evaluate the exact areas of open chromatin versus levels of RNA expression in any individual cell. Within a sample, this can allow greater extrapolation to trends and differences between cell types or even within a particular cell type.”
Protein-DNA Interactions
A number of key epigenetic sequencing methods are used for profiling important protein-DNA interactions. These types of epigenomic mapping can reveal “the location and abundance of specific proteins on chromatin, from histone modifications to transcription factors and chromatin regulations,” explained Martis W. Cowles, Ph.D., Chief Business Officer at EpiCypher. “This provides key insights into gene regulation and can help us to understand important ‘non-coding’ regions of the genome, such as promoters and enhancers. Studying the proteins that interact with chromatin to turn genes on and off can help us understand what drives changes in gene expression in response to stimulus, drug treatment, and/or in disease.”
ChIP-Seq
One of the most traditional techniques for mapping protein-DNA interactions is ChIP-Seq (chromatin immunoprecipitation followed by sequencing). “It is similar to some array-based technologies; however, it produces data with better resolution, less noise, and higher coverage than array-based technologies,” explained O’Hara. “Similar to Methyl-Seq, this method typically employs the comparison of a sample versus a control sample.”
Performing ChIP-Seq begins with the fixation of cells to maintain protein-DNA interactions at their native sites. Then the cells are lysed and the chromatin is fragmented to its desired length. After fragmentation, the target of interest is bound using a specific antibody that assists in the isolation of the chromatin. These isolated regions are then converted into sequencing libraries before being loaded onto the appropriate instrument. The resulting data is mapped back to the genome to uncover any significant DNA interactions.
Figure 1: Brief overview of several epigenetic methods reviewed in this article (courtesy of Azenta Life Sciences)
“ChIP-Seq is an excellent exploratory tool as it can be used to learn about many different types of modifications, but does require many controls to accurately interpret results and confirm antibody specificity,” stated O’Hara. While ChIP-Seq is still commonly used in epigenetics research, several newer methods have expanded the way that scientists investigate these interactions.
CUT&RUN and CUT&Tag
CUT&RUN (cleavage under targets & release using nuclease) and CUT&Tag (cleavage under targets and tagmentation) technologies are more recent and innovative approaches to performing these types of epigenetic mapping. Cowles explained that due to their sensitivity and throughput, they’re “game changers” when compared to existing ChIP-Seq-based approaches. “CUT&RUN and CUT&Tag are compatible with as few as 5,000 cells, which is a >100-fold improvement over ChIP-Seq. Further, these assays don’t require chromatin fragmentation, allowing the workflows to be easily automated for scaled clinical studies.”
CUT&RUN is a method that relies on the targeted cleavage of protein-DNA complexes by a protein A/G-fused micrococcal nuclease (pAG-MNase). The process begins by immobilizing cells and then using antibodies specific to the protein of interest as guides for the localization of pAG-MNase to the target site. The protein-DNA complexes are then released after cleavage and the DNA fragments are purified, sequenced, and analyzed to identify the protein-binding sites in the genome.
CUT&Tag utilizes a similar principle but includes the use of a Tn5 transposase fused to protein A. Using antibodies that bind to the target of interest (e.g., a chromatin post-translational modification), the fusion protein is directed to the target site in the chromatin, and the Tn5 transposase simultaneously cleaves and adds sequencing adapters to the DNA fragments. This allows for subsequent sequencing and analysis to identify histone modifications or the binding sites of specific proteins.
Both of these methods have a wide range of applications. “Many researchers are using CUT&RUN and/or CUT&Tag to study the distribution and abundance of their favorite histone modification, transcription factor, or chromatin regulator, and then studying how these factors change in response to cellular stimulation, drug treatment, and/or disease,” said Cowles. “Our pharmaceutical partners are also using these technologies to study drug mechanisms of action as well as identify epigenomic biomarkers to monitor disease and treatment response.”
While antibody selection has traditionally been a challenge for these methods, Cowles explained that recent advancements include the use of DNA-barcoded nucleosomes as spike-in controls. “We originally developed these “SNAP” spike-in controls for ChIP-Seq (SNAP-ChIP®), and have since updated them for compatibility with CUT&RUN and CUT&Tag (SNAP-CUTANA®). EpiCypher’s SNAP spike-in controls are valuable tools to identify high-performing antibodies and take the mystery out of assay optimization so that you can trust your data.”