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Pore-C: multi-contact, chromosome conformation capture for both genome-wide and targeted analyses


Date: 3rd December 2020

Higher-order chromatin structure arises from physical interactions of many genomic loci. Pore-C investigates this aspect of genome architecture by directly sequencing multi-way chromatin contacts

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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 to fit inside a nucleus, but must remain accessible for gene transcription, replication and repair. Control elements and their target genes are not always adjacent in the linear sequence, and so folding is not random. Pore-C explores the folded state of the genome, which can tell us about genome function and regulation. 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). Nanopore reads span entire amplicons, which can contain fragments from multiple interacting loci (Fig. 1b). Each segment of the read is assigned to a restriction fragment, determined by 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). We have released Pore-C sample preparation protocols for cells grown in culture, insects, nematode worms, mammalian blood and tissue and plants.

Fig. 2 Pore-C a) contacts b) flow cell yields c) different restriction enzymes d) Hi-C comparison

Performance statistics for Pore-C datasets derived from human cell lines

We prepared Pore-C libraries from cultured human cell line samples containing 5 million total cells each. This generated approximately 25 million reads per PromethION flow cell, resulting in 510 million total Pore-C contacts and 177 million contacts (Fig. 3a). Cumulative yield over time approaches 80 Gb, with read N50s of 4-5 kb, and the longest reads in excess of 10 kb (Fig. 3b). The choice of restriction enzyme in the digestion step influences both the fragment length and the total number of contacts. Both are highest using NlaIII (Fig. 3c). By calculating the number of pairwise contacts generated per Gb sequenced, head to head comparisons can be made with Hi-C, showing that Pore-C using NlaIII can output roughly as many contacts per Gb sequenced as 100 bp paired-end Illumina Hi-C reads (Fig. 3d).

Fig. 3 Targeted Pore-C a) workflow b) performance c) coverage d) TAD disruption

Disrupted regulatory mechanisms in Fragile X Syndrome can be seen with targeted Pore-C

Fragile X Syndrome (FXS) is caused by a trinucleotide CGG tandem repeat expansion in the 5’ UTR of the FMR1 gene, which is located at the boundary of two topologically associated domains (TADs). The disease-associated repeat expansion disrupts the 3D chromatin structure of the surrounding region. We combined Pore-C with hybrid capture to enrich a 2.2 Mb region encompassing the FMR1 TADs (Fig. 4a). Targeted Pore-C increases the number of on-target contacts ~10-fold, with up to 2 million on-target contacts per PromethION (TM) Flow Cell (Fig. 4b). We obtained~500x coverage of patient and control cells from a single PromethION Flow Cell (Fig. 4c). Depletion of contacts is seen in HG002 cells (Fig. 4d red box) representing the normal FMR1 TAD boundary. This is structurally disrupted by the disease-associated repeat (Fig. 4d, lower).

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