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Pore-C: nanopore sequencing of DNA concatemers reveals higher-order features of chromatin structure


Date: 5th December 2019

Imielinski lab (NYGC) collaboration: probing the three-dimensional spatial organisation of chromatin in human cells using a combination of long-read sequencing and chromatin-conformation capture 

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Fig. 1 Pore-C a) laboratory workflow b) multi-contact reads c) overview of bioinformatics workflow d) good concordance between Hi-C pairwise and Pore-C virtual pairwise datasets

Exploring the three-dimensional state of the human genome and detecting multi-way, long- range interactions using chromatin conformation capture and nanopore sequencing

Genomic DNA must be folded in order to fit inside a nucleus, and yet the DNA must remain accessible for gene transcription, DNA replication and repair. Control elements and their target genes are not always adjacent in the linear sequence, and folding is therefore not random. The aim of Pore-C is to explore the folded state of the genome, which can tell us about genome function and regulation. In the Pore-C protocol, genomic DNA is first cross-linked to histones, preserving the spatial proximity of interacting loci. Restriction digestion followed by proximity ligation is used to join cross-linked, interacting fragments, which are then sequenced (Fig. 1a). Long nanopore reads span entire amplicons, which can contain fragments from multiple interacting loci (Fig. 1b). Reads are aligned to a reference sequence to identify separate alignments and each segment of the read is assigned to a restriction fragment, determined through in silico digestion of the reference sequence. The reference genome is then divided into equally sized bins and restriction fragments are assigned to their corresponding bin. Finally, the total number of bin-to-bin contacts is calculated from all reads and visualized in a contact map (Fig. 1c). When the Pore-C reads are simplified to a set of virtual pairwise contacts, the data is concordant with Hi-C contacts at the chromosome and territory level (Fig. 1d).

Fig. 2 Concordance of Pore-C virtual pairwise contact maps with Hi-C data at a-c) compartment d-f) TAD and g-j) loop levels and k) analysis of high-order long-range contacts

Pore-C-derived contact maps can be used to identify standard features of chromatin architecture and reveal higher-order contacts, reflecting combinatorial chromatin states

To compare Pore-C data to the previously-published “gold standard” GM12878 Hi-C dataset (4D Nucleome project, Rao et al., Cell 159, 2014) at greater levels of resolution, we constructed a contact map using NlaIII-derived virtual pairwise Pore-C contacts. Good correlation was obtained between Pore-C and Hi-C data when identifying compartments (Figs. 2a-c), topically associating domains (TADs, Figs. 2d-f) and loops (Figs. 2g-j). From these comparisons we can conclude that there is high concordance between the probability of two sequences sharing a Pore-C concatemer and their Hi-C pairwise contact probability, that Pore-C DNA concatemers reflect known, previously characterized pairwise features of chromatin architecture and that the Pore-C protocol represents a scalable alternative to a standard Hi-C experiment. A key additional feature of Pore-C is its ability to detect high-order chromatin structure. To explore biological structures revealed by high-order and long-range (HOLR) contacts, we studied the combinatorial connectivity within and between A (active) and B (inactive) compartments revealed by both Pore-C and SPRITE, a previously published higher-order chromatin profiling technology (Quinodoz et al. Cell 174, 2018). We found that Pore-C HOLR contacts reflect direct molecular interactions more accurately than their SPRITE counterparts (Fig. 2k). Additionally, Pore-C can resolve structure in repetitive genomic regions that are unmappable and hence invisible to the short-read SPRITE map.

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