Biological insights with Pore-C: investigating the spatial organisation of chromatin in the human genome

We generated contact data for human breast cancer cell line HCC1954 and control sample ANA51. Using the tool JaBbA we identified a complex, multi-chromosomal subgraph connecting chromosomes 9, 12 and 20 in HCC1954 (Fig. 1a). We obtained a genome graph by analysis of read-depth and junction calls. Higher-order contacts mapping to the region were used to nominate an amplified multi-junction derivative allele within the graph. The Pore-C alignments and associated contact maps visually confirm this putative derivative allele. For additional confirmation we designed multi-colour FISH probes to target locations on the allele (Fig. 1b). Co-  localisation of probes in the HCC1954 metaphase spreads confirm the presence of an amplified multi-junction fusion of chromosomes 9, 12 and 20 (Fig. 1c) which is not seen in ANA51.

Assembly contiguity can be improved substantially by the addition of relatively low coverage of Pore-C data using the pipeline shown in Fig. 2a. We generated approximately 130 Gb of reads from the human genome NA24385 using one PromethION flowcell and assembled these using redbean. The resulting assembly had an NG50 of 10.4 Mb. The inclusion of an additional flowcell of Pore-C reads increased the assembly contiguity substantially, with reads produced by HindIII giving the greatest increase, to 98.6 Mb (Fig. 2b). Prior to scaffolding, when Pore-C reads are plotted against the redbean contigs, many off-diagonal features are visible, indicating suboptimal assembly (Fig. 2c, shown for Chr. 4). Following Pore-C scaffolding, a more optimal assembly is obtained, with just three scaffolds covering the entire chromosome (Figs. 2d and 2e).

To compensate for the presence of an additional copy of the X chromosome in females (XX) compared to males (XY), a random haplotype of the female X chromosomes is silenced through an epigenetic process known as X-chromosome inactivation (XCI). We made use of the publicly available chromosome-level haplotype information for the human cell line GM12878 to separate NlaIII- derived Pore-C reads according to whether they originated from the inactive (Xi) or active (Xa) X chromosome and to thereby detect differences in the DNA conformation and methylation state between the two haplotypes. Imaging studies have shown that Xi adopts a distinctive structural conformation known as the Barr body. 3C methods have shown that this condensed structure is in fact bipartite, consisting of two superdomains separated by a hinge region centered on the macrosatellite repeat locus DXZ4. Xa on the other hand has a more typical elongated structure with A/B compartments (Fig. 3a). Multiple layers of transcriptional repression ensure that the majority of the approximately 1,000 genes on Xi are not expressed, but ~20% of these genes can ‘escape’ this repression and are characterised by DNA methylation patterns more typical of genes on Xa (Fig. 3b). Allele-specific chromatin contact maps at 500 kb resolution (Fig. 3c) for Xi (above the diagonal) and Xa (below the diagonal) show Xi-specific superdomains as two triangles above the diagonal that touch at the DXZ4 region, while the checkerboard pattern associated with A/B compartmentalisation is visible on Xa. Methylation levels detected using Pore-C reads at CpG islands within 1 kb of transcription start sites confirms the pattern of differential methylation at loci subject to XCI. In contrast, similar levels of methylation are found at loci that escape XCI (Fig. 3d). A positional analysis of the Pore-C-derived methylation signal shows a high level of differential methylation around the promoter region for XCI loci but not for those that escape XCI (Fig. 3e).

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