Chromatin conformation capture (3C) techniques reveal genomic interactions in three dimensions. This can provide key information on the effect of chromatin structure on transcriptional regulation; the data can also be utilised to orient contigs, producing highly contiguous scaffolded assemblies. However, the traditionally used short-read 3C methodology limits the number of contacts available to analyse per read. Combining chromatin conformation capture with long-read nanopore sequencing, Pore-C provides long-range contact information, shedding light on higher-order structure. The technique is PCR-free, allowing the characterisation of base modifications in the same dataset.
- Use long sequencing reads to generate enhanced, multi-way chromatin interaction data
- Generate new insights into gene expression, enhance genome assembly contiguity, and investigate modified bases
- Pore-C offers a complete, end-to-end chromosome conformation capture workflow — from sample to results
A complete, end-to-end workflow for chromosome conformation capture
Oxford Nanopore provides the complete solution for investigating chromatin conformation — Pore-C, an end-to-end workflow, from sample preparation to analysis. Long nanopore sequencing reads enable long-range, multi-way contact information; this cannot be achieved using Hi-C — the commonly used short-read-based 3C method, which produces pairwise data, limiting its utility for generating higher-order structural information. Furthermore, Pore-C does not require amplification, enabling direct detection of modifications alongside nucleotide sequence in the same dataset — for an even more comprehensive understanding of the regulation of gene expression.
Enhance genome assemblies using long-range Pore-C data
As well as investigating chromatin architecture, chromatin contact information from Pore-C can be used to scaffold existing genome assemblies to high contiguity, and is an extremely effective tool for phasing assembly graphs (Figure 2), enabling haplotype-resolved telomere-to-telomere (T2T) assemblies, even in the absence of parental sequencing data. T2T assemblies unlock the most complex genomic regions, and are now possible to achieve using one sequencing platform — Oxford Nanopore duplex, ultra-long, and Pore-C sequencing reads enable the assembly of accurate and complete reference genomes. The data also allows resolution of entire megabase-sized structural variants (SVs), and an understanding of their association with chromatin conformation (see case study below).
From the same sample, and the same sequencing run, the following can therefore be obtained with Pore-C:
- Higher-order, multi-way contact data
- Epigenetic modifications
- Whole-genome sequence data for downstream assembly
- SV detection and resolution — including highly-complex, megabase-spanning variants
Together, these provide an unprecedented understanding of the regulation of gene expression.
Obtaining higher-order features of chromatin structure
Deshpande et al. investigated high-order genome structure in human cells with Pore-C, to gain a greater understanding of complex cancer genomic rearrangements and improve assembly scaffolding. The team determined that Pore-C assesses high-order contacts with substantially greater efficiency than other approaches. As nanopore sequencing of native DNA does not require PCR, they were also able to determine the methylation status of DNA participating in such high-order interactions relative to pairwise interactions occurring at the same loci.
Phasing human genomes using long nanopore sequencing reads
Phasing human genomes is important across clinical and life sciences research. Lorig-Roach et al. used ultra-long nanopore reads (>100 kb) in the Pore-C workflow — generating more contact data than an alternative long-read sequencing platform, which can only use size-selected reads of 10–30 kb — to generate highly-contiguous, phased, nanopore-only human genome assemblies.
View the Pore-C protocols
Get detailed information on Pore-C sample preparation and sequencing.
How can I investigate chromatin conformation with Oxford Nanopore?
For a comprehensive investigation into chromatin conformation with Oxford Nanopore sequencing technology, we recommend following the Pore-C extraction and library preparation protocol, then sequencing your library on a single PromethION Flow Cell as a starting point. Depending on the resolution desired, additional sequencing runs can be performed. The Pore-C info sheet provides advice regarding cell number and restriction enzyme usage, as well as other considerations that need to be made prior to commencing your Pore-C experiment.
A wide range of Pore-C sample preparation and sequencing protocols are available on the Extraction Protocols page of the Prepare Documentation section of the Nanopore Community website.
For information on generating highly contiguous, chromosome-scale plant genome assemblies, view the complete workflow, featuring recommendations from sample preparation right through to data analysis.
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