Applications Investigations
Epigenetics
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 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 detected via long-read sequencing — with no additional library prep steps. Long-range epigenetic changes, structural variants, and single nucleotide polymorphisms can be characterised in a single dataset.
Detect base modifications alongside nucleotide sequence as standard
Phase epigenetic DNA modifications or unambiguously assign RNA modifications to transcript isoforms using long sequencing reads
Use whole genome or amplification-free targeted sequencing approaches
INTRODUCTION
Detect a wide range of base modifications alongside nucleotide sequence
Using nanopore sequencing, researchers have directly identified DNA base modifications at nucleotide resolution, including 5mC (Figure 1), 5hmC, m6A, and BrdU, with detection of other natural or synthetic epigenetic modifications possible through training basecalling algorithms. Base modifications can be detected across the entire genome, or, by using amplification-free CRISPR/Cas-based enrichment, targeted genomic regions (Figure 1). The use of direct nanopore sequencing technology means that even if base modifications are not a primary objective of your DNA sequencing study, the data is available for analysis at any future timepoint.
Figure 1: Both hypermethylation and repeat expansion are found in intron 1 of the frataxin (FXN) locus in Friedreich’s ataxia, a recessive neurodegenerative movement disorder. Targeted, amplification-free nanopore sequencing using CRISPR/Cas9 enrichment enabled identification of repeat length (GAA triplet expansion) and methylation (5mC) status of the affected locus in a family trio (mother, father, child). Oxford Nanopore Technologies products are currently for research use only.
Figure 2: The modified base detection tool Tombo was applied to NA12878 genomic data generated using PromethION. The top two panels show methylation at a CpG site, which is symmetric on positive and negative strands. The bottom two panels show methylation in examples of CHG and CHH contexts. Here the methylation was asymmetric, only being present on the positive strands. Data was highly correlative with that obtained using bisulfite sequencing. Raw nanopore signals (red lines); expected canonical levels (grey background distributions).
Assign base modifications to specific RNA isoforms using full-length reads
Nanopore sequencing is the only currently available technology that allows direct sequencing of native RNA, with no requirement for amplification or reverse transcription. As a result, base modifications are preserved and can be detected alongside the RNA sequence. Furthermore, long nanopore reads allow full-length sequencing of transcripts and long non-coding RNA (lncRNA) — enabling unambiguous assignment of modifications to specific RNA isoforms. A number of methylation-aware basecallers and analysis tools are now available, including Tombo (Figure 2). In addition to the identification of a number pre-determined base modifications, Tombo can be trained to detect additional non-standard bases.
CASE STUDY
Detecting imprinted DNA methylation
‘We demonstrate that long-read sequencing using nanopore technology can efficiently generate haplotyped mammalian methylomes’
Gigante et al.Methylation of cytosines at CpG islands is known to play a key role in embryonic development. It is also responsible for the phenomenon of genomic imprinting, whereby genes are expressed according to the parent of origin. The study of allele-specific methylation using short-read sequencing technology is hampered by the requirement for high SNP density (for haplotyping), the inefficient bisulfite sequencing workflow, and the potential for PCR bias. Using long and direct nanopore sequencing reads, Gigante et al. were able to overcome all of these challenges to determine allele-specific methylation in mouse placenta cells. With ~10x genomic coverage, the team were able to confirm 70 previously identified imprinted genes and propose a further 65 candidates. Due to their long length, 75% of nanopore sequencing reads could be haplotyped.
Visit the Protocol builder to create a custom protocol for your epigenetics sequencing project.
SEQUENCING WORKFLOW
How do I detect base modifications using nanopore sequencing?
MinION and GridION devices are ideal for epigenetic analysis of microbial genomes or eukaryotic transcriptomes and targeted genomic regions, while PromethION is suitable for high-throughput epigenetic studies of larger genomes. Amplification-free library preparation is required in order to maintain base modifications. For DNA sequencing, we recommend the Ligation Sequencing Kit, while for RNA sequencing, the Direct RNA Sequencing Kit is required. To identify modified bases, a number of Oxford Nanopore- and Community-developed basecallers are available, including Guppy, Tombo, Megalodon, and Nanopolish. The choice of which is highly dependent upon your experimental aims. The integrated Guppy base caller enables direct detection of 5mC and m6A modifications as standard. Tombo is one of the most flexible basecallers, detecting a number of common base modifications and capable of being trained to detect additional, non-standard bases.
GET STARTED
Genome-wide detection of base modifications in plants and animals
For whole-genome detection of modified bases in plants and animals, we recommend the following:
Ligation Sequencing Kit (SQK-LSK109)
Ligation Sequencing Kit (SQK-LSK109)
Analysing microbial base modifications or performing amplification-free target enrichment? Find out more about our MinION and GridION devices.
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