Pore-C: a complete, end-to-end workflow for chromatin conformation capture, published in Nature Biotechnology

In a recently published paper, Oxford Nanopore’s Genomic Applications team, in collaboration with Marcin Imielinski’s lab at Cornell/NYGC, outline their complete solution for investigating chromatin conformation

Pore-C, an end-to-end workflow unique to Oxford Nanopore, combines chromatin conformation capture (3C) with direct, long nanopore sequencing reads. With nanopore reads, long-range, multi-way contact information can be obtained. 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.

Investigating genome architecture and enabling large genome assembly

This newly published workflow not only reveals higher-order contact information from multiple loci, but also large structural variants and chromosomal rearrangements. Furthermore, Pore-C requires no amplification steps, so repetitive and GC-rich regions are resolved with high contiguity and DNA modifications are preserved in the long nanopore reads. Epigenetic and higher-order contact information enables genome-wide analyses of 3D cooperativity.

This means that Pore-C can be used to scaffold and improve the quality of existing genome assemblies to generate chromosome-scale assemblies, discover unique insights into the higher-order genome organisation of a species of interest, and reveal epigenetic information for a more comprehensive understanding of gene regulation. For example, gene activation, or silencing, may require the interaction of two or more physically distinct loci. Pore-C can detect such higher-order chromatin complexes, revealing enhancers, silencers, and insulators in one long sequencing read.

More in-depth interrogation

Using Oxford Nanopore sequencing the following can be obtained with Pore-C from the same sample, and the same sequencing run:

• Higher-order, multi-way contact data

• Epigenetic modifications

• Proximity information which enables chromosome-scale scaffolding and phasing

• Structural variation detection and resolution — including highly-complex, megabase-spanning variants.

Together, these provide an unprecedented understanding of the regulation of gene expression, all on one platform.

The Pore-C workflow has a wide range of potential uses, including cancer research. In the study Deshpande et al. explain that ‘Pore-C profiling of malignant cells may help identify... noncoding structural variant cancer drivers, which may be particularly relevant to the biology of advanced and therapy-resistant cancers’. Additionally, when used with Oxford Nanopore’s highest-throughput PromethION device, the study team generated up to 200 Gb of Pore-C sequence data with ‘comparable per Gb reagent costs’ to standard 3C methodologies.

Pore-C allows us to fully explore the complexities of long-range genomic and epigenomic interactions across the 3-dimensional genome. Additionally, Pore-C could enhance large genome assemblies for a vast range of organisms, from animals to plants and even those that are polyploid or have completely unknown genome sequences. This has the potential to give us a far deeper understanding of genomic organisation as well as cellular processes such as gene regulation.

Pore-C links:

• Deshpande, A.S., Ulahannan, N., Pendleton, M. et al. Identifying synergistic high-order 3D chromatin conformations from genome-scale nanopore concatemer sequencing. Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01289-z: https://www.nature.com/articles/s41587-022-01289-z ($)

• Specific Pore-C analysis pipelines are readily available on GitHub. Visit the Nanopore Community for guidance through every step of the Pore-C workflow: https://community.nanoporetech.com/info_sheets/restriction-enzyme-pore-c/v/rpc_s1015_v1_revf_12nov2019

• Optimised Nanopore workflows have been developed: blood samples, plants, and cell culture, animal, or insect tissues – the method is simple and scalable.  The plant Pore-C workflow https://nanoporetech.com/sites/default/files/s3/literature/plant-pore-c-workflow.pdf

• Pore-C: multi-contact, chromosome conformation capture for both genome-wide and targeted analyses (poster): https://nanoporetech.com/resource-centre/porec