Epigenetics sequencing and analysis with nanopore technology
Epigenetics, the study of heritable phenotypic changes that do not involve alteration of the DNA sequence, has made significant progress over the last decade due to advancements in high-throughput, high-resolution analysis techniques, and many human diseases are now known to have an epigenetic component. Delivering long, direct sequencing reads, nanopore technology provides new opportunities for epigenetics research, including simultaneous detection of nucleotide sequence and DNA and RNA base modifications, complete characterisation of lncRNAs, and more comprehensive chromatin capture studies — providing new insights into this exciting field of study.
- Simultaneously call nucleotide sequence and base modifications in the same sequencing run — no protocol adaptations, chemical conversion, or additional sequencing required
- Phase epigenetic DNA modifications or unambiguously assign RNA modifications to transcript isoforms using long sequencing reads
- Comprehensively characterise full-length lncRNAs using long sequencing reads
- Identify more chromatin interactions and detect modified bases using long, direct sequencing reads
- Analyse data using a growing number of tools
- Scale to your needs using Flongle, MinION, GridION, or PromethION
How will you use nanopore technology?
Long non-coding RNA (lncRNA)
Chromosome conformation capture
Cost-effectively and simultaneously detect epigenetic DNA modifications alongside nucleotide sequence — with no requirement for chemical conversion or additional sequencing. Nanopore technology enables direct sequencing of native DNA molecules, eliminating the requirement for amplification and preserving epigenetic modifications. Researchers have utilised nanopore sequencing to detect 5mC, 5hmC, m6A, and BrdU, with detection of other natural or synthetic epigenetic modifications possible through training basecalling algorithms.
- Investigate the role of epigenetic modifications in disease and identify potential prognostic and diagnostic indicators
- Explore the role of epigenetic modifications in microbes and microbial communities
- Elucidate DNA replication dynamics using synthetic base analogues
- Streamline your workflow — rapid 10-minute library prep and no bisulfite conversion required
- Use whole genome, or amplification-free targeted sequencing techniques
- Train basecalling algorithms to detect any base modification
‘Methylation data can directly be obtained from the same WGS data set which makes time-consuming bisulfite conversion and specialized methylation assays (sequencing or hybridization-based) expendable’
Targeted nanopore sequencing with Cas9 for studies of methylation, structural variants and mutations
With over 170 known forms, epigenetic RNA modifications are highly abundant and play an important role in RNA stability, localisation, and protein translation. Many RNA modifications have been linked to human disease, such as cancer and neurological disorders. Nanopore technology allows direct detection of epigenetic RNA modifications alongside nucleotide sequence — with no chemical or protocol adaptations. Long, full-length reads enable unambiguous assignment of modifications to specific transcript isoforms, opening new avenues in epigenetics research.
- Identify RNA modifications directly, alongside nucleotide sequence
- Accurately assign epigenetic modifications to specific transcript isoforms using, long, full-length sequencing reads
- Rapid, streamlined protocol with no harsh or inefficient chemical adaptations required
- Train basecalling algorithms to detect novel epigenetic modifications and synthetic base analogues
‘Direct RNA methylation analysis will radically change how people study RNA in the next decade’
Accurate detection of of m6A RNA modifications in native RNA sequences using third-generation sequencing
Long non-coding RNAs (lncRNAs) are RNA molecules that are over 200 nucleotides long and which do not encode proteins. These molecules regulate gene expression and, in humans, are over four times more abundant than coding RNA sequences. The long sequencing reads generated by nanopore technology allow full-length sequencing of lncRNA molecules in single reads, allowing comprehensive characterisation of these important but poorly understood epigenetic molecules.
- Unambiguously characterise lncRNAs in single, full-length reads
- Eliminate PCR bias using direct cDNA or direct RNA sequencing
- Direct sequencing of native RNA offers potential for simultaneous detection of epigenetic base modifications alongside nucleotide sequence
- Get higher yields from less input using updated RNA and cDNA sequencing kits
‘Nanopore has a range of advantages over other sequencing approaches. By dispensing with the amplification or enzymatic modification of target molecules, important sources of bias are avoided. cDNA molecules can be directly sequenced with minimal preparation, and it may even be feasible to identify chemically modified bases’
London Calling 2019: Sílvia Carbonell Sala lightning talk
The long, direct reads and scalable, high-throughput sequencing capability delivered by nanopore technology, allows more detailed insight into chromosome conformation than provided by existing short-read 4C, 5C, or Hi-C sequencing approaches.
- Simple, streamlined workflow
- Optional PCR-free approach enables detection of epigenetic DNA base modifications alongside nucleotide sequence
- Visualise higher order interactions using long sequencing reads
- Sequence across repetitive regions
- Scale your research using Flongle, MinION, GridION, and PromethION platforms
‘Potential advantages of Pore-C are…simpler sample prep,…simultaneous readout of DNA modifications and contact probabilities, and higher order interactions with multi-way contacts’
London Calling 2019: Eoghan Harrington