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
Pore-C offers a complete, end-to-end chromosome conformation capture workflow — from sample to results
Generate new insights into gene expression, investigate modified bases, and enhance genome assembly contiguity
A complete, end-to-end workflow for chromosome conformation capture
Oxford Nanopore provides the complete solution for investigating chromatin conformation: Pore-C is an end-to-end workflow, from sample preparation to analysis. Pore-C is provided as a comprehensive sample preparation and sequencing protocol (see the Extraction methods section of the Knowledge page on the Community), and an analysis pipeline available in GitHub (Figure 1).
With long nanopore sequencing reads, you can obtain 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 requires no amplification step. Therefore, combined with the direct detection of modifications alongside nucleotide sequence, you can achieve an even more comprehensive understanding of the regulation of gene expression.
Enhanced genome assemblies using long-range Pore-C data
As well as investigating chromatin architecture, the long reads obtained from the Pore-C workflow enable users to scaffold genome assemblies and correct genomes. Assembly contiguity can be substantially improved with the inclusion of Pore-C data (Figure 2). The Oxford Nanopore GitHub Pore-C analysis pipeline includes an assembly and scaffolding workflow, in addition to processing and outputting pairwise contact data. The data also allow 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
‘...our results establish Pore-C as the most simple and scalable assay for the genome-wide assessment of combinatorial chromatin interactions, with additional applications for cancer rearrangement reconstruction and de novo genome assembly’
Ulahannan et al. investigated chromatin structure in human cells with Pore-C, using this technique to additionally gain a greater understanding of complex cancer genomic rearrangements and improve assembly scaffolding. The team determined that Pore-C higher-order contacts, decomposed into pairwise contacts, reflected previously characterised pairwise interactions obtained using Hi-C. In addition, in the breast cancer cell line HCC1954, Pore-C data identified the presence of a complex amplification and rearrangement, connecting chromosomes 9, 12, and 20. This result was confirmed using fluorescence in situ hybridisation (Figure 3). Finally, the team demonstrated that Pore-C improves the quality of de novo genome assembly.
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 protocol, and 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.
The Pore-C sample preparation and sequencing protocol is available on the Extraction methods page of the Knowledge section of the Nanopore Community website.
We recommend following one of the two Pore-C pipelines from the Oxford Nanopore GitHub resource for analysis of your Pore-C data:
- Pore-C-Snakemake: a more comprehensive analysis pipeline — recommended. This pipeline also aligns reads against the genome and converts pairwise contacts to contact matrices, enabling you to then view the data as a contact map (as seen above in Figure 1c).
- Pore-C: analysis of Pore-C sequencing data, adapted to deal with multi-contact reads. This pipeline deconstructs the multiway contacts into pairwise contacts in standard format (.pairs, .bed), for downstream analysis.
Note: both analysis pipelines require familiarity with the command line. Further advice and support can be found in the Nanopore Community.
Chromosome conformation capture: in action
To perform Pore-C chromosome conformation capture experiments, we recommend the following: