Epigenetics and methylation analysis
Epigenetics, the study of heritable phenotypic changes that do not involve alteration of the nucleotide sequence, plays a key role in gene expression and has been associated with many diseases, including cancer. As PCR removes base modifications, their detection via traditional sequencing technologies requires the use of special library preparation steps (e.g. bisulfite conversion) which damage nucleic acids, resulting in very short sequencing reads. With nanopore sequencing, PCR is not required, enabling DNA and RNA modifications to be preserved and directly sequenced — with no additional library prep steps. Long-range epigenetic changes, structural variants, and single nucleotide polymorphisms can be identified and phased in a single dataset.
Introduction
Directly detect DNA and RNA methylation with high reproducibility and low bias
Using nanopore sequencing, researchers have directly identified DNA and RNA base modifications at nucleotide resolution, including 5mC, 5hmC, 6mA, and BrdU in DNA, and m6A in RNA, with detection of other natural or synthetic epigenetic modifications possible through training basecalling algorithms.
One of the most widespread genomic modifications is 5-methylcytosine (5mC), which most frequently occurs at CpG dinucleotides. Compared to whole-genome bisulfite sequencing, the traditional method of 5mC detection, nanopore technology calls a higher number of CpG positions in the genome, requires less sequencing data, and shows more even genomic coverage with considerably lower GC bias; analysis runtime is also significantly shorter (Figure 1).
Benefits of nanopore technology over traditional bisulfite sequencing for methylation analysis:
- More even genomic coverage with lower GC bias
- Higher number of CpG positions called at lower read depth
- Haplotype phasing of methylated bases using long reads
- Greater experimental reproducibility
- Considerably faster data analysis
Figure 1: Benchmarking of 5mC DNA methylation calling was performed with genomic data from two replicates of the NA12878 human genome sequenced on a single PromethION™ Flow Cell, to obtain 20x depth of coverage per sample. DNA methylation calling was compared to two 50x short-read whole-genome bisulfite sequencing datasets (from ENCODE and GEO), using the Megalodon tool developed by Oxford Nanopore (a). Compared to the bisulfite sequencing method, nanopore technology shows: greater uniformity in mapping (b), significantly less impact of GC bias (c), lower read depth requirement (d-f), greater reproducibility (g), and faster analysis (h). Note: the Megalodon tool has been superseded by Remora.
Figure 2: Long nanopore reads and even depth of coverage allows accurate, long-range, haplotype-resolved DNA methylation calling. Differential methylation at a known imprinted genomic region, containing genes BCAP31 and ABCD1, was identified in the methylation benchmarking data (NA12878) described in Figure 1.
Accurately phase methylation with long nanopore reads
Both hypermethylation and hypomethylation of 5mC at CpG dinucleotides have been shown to be associated with diseases. With nanopore technology, such differential methylation can be identified and phased into haplotypes with nucleotide-level precision (Figure 2).
Methylation and other natural or synthetic base modifications can be detected across the entire genome, or, via amplification-free CRISPR/Cas-based enrichment, at targeted genomic regions. The use of direct sequencing means that even if base modifications are not a primary objective of your DNA (or RNA) sequencing study, the data is available for analysis at any future timepoint.
Case study
Detecting methylation in repetitive regions of the human genome
‘we are just scratching the surface about unveiling epigenetic control of these [repetitive] regions’
With the limited capacity of traditional short-read sequencing technologies to access repeat-rich regions, such areas of the human genome have remained underexplored. Ariel Gershman and colleagues at Johns Hopkins University, USA, have been using long nanopore reads to reveal the methylome of previously unexplored large repetitive arrays in the human genome. Analysis of higher order repeats (HORs) at chromosome centromeres, which provide binding sites for the centromere-associated histone variant CENPA, uncovered distinctive patterns of hypomethylation and hypermethylation. Her team demonstrated for the first time how long nanopore reads could reveal long-range, allele-specific DNA methylation patterns across HORs and other classes of satellite repeats. These analyses have the potential to advance our understanding of the role of methylation in chromosome segregation and its association with genetic disorders.
View the workflow for calling and phasing 5mC in the human genome, from DNA extraction and library prep through to nanopore sequencing and analysis.
Sequencing workflow
How do I detect base modifications using nanopore sequencing?
PromethION is suitable for high-throughput epigenetic studies of larger genomes, such as human and many plant genomes, whilst MinION and GridION devices are ideal for epigenetic analysis of microbial genomes or eukaryotic transcriptomes and targeted genomic regions. Amplification-free library preparation is required in order to maintain base modifications. For DNA sequencing, we recommend the Ligation Sequencing Kit. For RNA modification analysis, the Direct RNA Sequencing Kit is required. Nanopore sequencing is the only currently available technology that allows direct sequencing of native RNA, with no requirement for amplification or reverse transcription.
To identify modified bases, a number of Oxford Nanopore- and Community-developed tools are available, the choice of which depends upon your experimental aims. For a simple point-and-click approach, 5mC and m6A analysis is integrated into MinKNOW, the software onboard nanopore sequencing devices. For best performance in 5mC methylation calling in the human genome, we recommend the analysis tool Remora.
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Genome-wide detection of DNA / RNA methylation in plants and animals
For whole-genome detection of modified bases in plants and animals, we recommend the following:
Analysing microbial base modifications or performing amplification-free target enrichment? Find out more about our MinION and GridION devices.
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