Digestion-ligation-only Hi-C is an efficient and cost-effective method for chromosome conformation capture

Abstract

Chromosome conformation capture (3C) technologies can be used to investigate 3D genomic structures. However, high background noise, high costs, and a lack of straightforward noise evaluation in current methods impede the advancement of 3D genomic research. Here we developed a simple digestion-ligation-only Hi-C (DLO Hi-C) technology to explore the 3D landscape of the genome. This method requires only two rounds of digestion and ligation, without the need for biotin labeling and pulldown. Non-ligated DNA was efficiently removed in a cost-effective step by purifying specific linker-ligated DNA fragments. Notably, random ligation could be quickly evaluated in an early quality-control step before sequencing. Moreover, an in situ version of DLO Hi-C using a four-cutter restriction enzyme has been developed. We applied DLO Hi-C to delineate the genomic architecture of THP-1 and K562 cells and uncovered chromosomal translocations. This technology may facilitate investigation of genomic organization, gene regulation, and (meta)genome assembly.

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Fig. 1: Flowchart of the digestion-ligation-only Hi-C (DLO Hi-C) method.
Fig. 2: The early quality-control step in the DLO Hi-C method.
Fig. 3: DLO Hi-C data analysis workflow and comparison of DLO Hi-C, in situ Hi-C, in situ DLO Hi-C, Hi-C, and DNase Hi-C for 3D genome structure analysis of K562 cells.
Fig. 4: Consistency of A/B compartments, TADs, and chromatin loops between DLO Hi-C, in situ DLO Hi-C, and in situ Hi-C.
Fig. 5: Detection of chromosomal translocations in THP-1 cells.

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Acknowledgements

We are thankful to Nature Research Editing Service for language editing. This work was supported by the National Key Research and Development Project of China (2016YFA0102500, to G.C.), the National Natural Science Foundation of China (grants 91440114, to G.L., and 31421064, to G.C.), Fundamental Research Funds for the Central Universities (grants 2662014PY001 and 2662015PY164, to G.C.), the National Key Research and Development Project of China (2017YFD0500303, to G.C.), the Huazhong Agricultural University Scientific & Technological Self-innovation Foundation (program 52204-13002, to G.C.), and the Huazhong Agricultural University Independent Scientific & Technological Innovation Foundation (program 2014bs13, to D.L.).

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Authors

Contributions

G.C., D.L., and G.L. contributed with conception of the project and experiment design. D.L. conducted the DLO Hi-C experiments and generated data. G.L., P.H., and W.X. performed data analysis and interpretation. D.L., S.Z., and Y.L. conducted the HCR experiment. D.L., P.H., G.L., G.C., L.L., K.Y., and M.J. wrote the manuscript, with input from all other authors. Z.F. and Y.R. revised the manuscript.

Corresponding authors

Correspondence to Guoliang Li or Gang Cao.

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Integrated supplementary information

Supplementary Figure 1 Half-linker ligation by T4 DNA ligase and T7 DNA ligase.

Unlike T4 DNA ligases, T7 DNA ligase cannot efficiently catalyze blunt-end ligation. Thus, after ligating the 20-bp MmeI half-linker by T7 DNA ligase, a 40-bp linker dimer is shown in the gel. T4 DNA ligase can ligate both blunt and sticky DNA ends, leading to a DNA smear in the gel. The experiment has been repeated four times independently with similar results.

Supplementary Figure 2 The structure of the 80-bp DLO Hi-C DNA fragment, Illumina sequence adapter, and PCR primers used in this study to construct the DLO Hi-C library.

a, The structure of the 80-bp DLO Hi-C DNA fragment, which contains a 40-bp complete linker and a pair of 20-bp interaction genomic sequences. b, The sequence of the Illumina sequence adaptor used to ligate the 80-bp DLO Hi-C DNA fragment. Because the overhang of the 80-bp DLO Hi-C DNA fragment is unknown, we designed a random 3′ cohesive end (5′-NN-3′) in the adaptor for ligation. c, The primers used to construct the DLO Hi-C DNA sequencing library are shown.

Supplementary Figure 3 Proof-of-principle experiment demonstrating the DLO Hi-C method.

a, K562 cells were double cross-linked with EGS and formaldehyde, digested with or without HindIII, and subjected to electrophoresis. b, The digested chromatin was ligated with or without the half-linkers by simultaneous digestion and ligation using T7 DNA ligase and HindIII and subjected to electrophoresis. c, Excessive linkers were removed by centrifugation. The linker-ligated chromatin complexes were precipitated after centrifugation, while the excessive MmeI linkers remained in the supernatant. d, Linker-ligated chromatin complexes were proximity ligated in gel by T4 DNA ligase, with the unligated sample (left lane) serving as a control. e, The proximity-ligated DNA was digested by MmeI to release the 80-bp DLO Hi-C DNA fragments, as indicated in the native PAGE image. f, Electrophoresis of the DLO Hi-C sequencing library. The experiment has been repeated three times independently with similar results.

Supplementary Figure 4 In situ DLO Hi-C experiment in K562 cells.

a, Violin plots showing the theoretical DNA fragment length distribution after digestion of human genome DNA by HindIII (n = 830,216), MboI (n = 7,067,604), and MseI (n = 18,882,492) (****P < 2.2 × 10–16, two-sided Wilcoxon rank-sum test). The box plot shows the first and third quartiles (top and bottom of boxes), the median (band inside the boxes), and the lowest and highest points within 1.5 times the interquartile range of the lower and higher quartile (whiskers). b, Nuclei were digested by MseI and subjected to electrophoresis. c, Verification of the integrity of the nuclei after proximity ligation by microscopy. d, Digestion of the in situ DLO Hi-C library by NheI for rapid early estimation of the portion of Hi-C ligation junctions. The experiments from bd have been repeated three times independently with similar results.

Supplementary Figure 5

Conformation and sequences of the full linkers.

Supplementary Figure 6 Delineation of the 3D genome structure of THP-1 cells.

a, Heat maps of THP-1 genome interactions reconstructed from the HindIII, BglII, and MseI datasets. b, Comparison of the topologically associated domains (TADs) of THP-1 chromosome 7 (from 8 Mb to 14 Mb) reconstructed from the HindIII, BglII, and MseI datasets. c, Venn diagram of the TAD boundaries from the HindIII, BglII, and MseI datasets.

Supplementary Figure 7 Density plot of normalized interchromosomal interaction counts.

The plot shows the smoothed density of normalized interaction counts (>0) between different chromosomes at a resolution of 1 Mb from the combined HindIII, BglII, and MseI datasets.

Supplementary Figure 8 New strong translocation sites in THP-1 cells identified by DLO Hi-C.

ad, The x and y axes show the regions of each chromosome at a resolution of 1 Mb, and the z axis reveals normalized contact counts of interchromosomal interactions from the combined HindIII, BglII, and MseI datasets.

Supplementary Figure 9 Expected mapping efficiency using different restriction enzymes.

ad, As the last base(s) of the restriction enzyme cutting site were removed during linker ligation, we re-added the lost base(s) back to the mapping tail. e, Plot of mapping efficiency and tail length. The x axis indicates the mapping tail length; The y axis indicates the ratio of uniquely mapping reads to total reads.

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Lin, D., Hong, P., Zhang, S. et al. Digestion-ligation-only Hi-C is an efficient and cost-effective method for chromosome conformation capture. Nat Genet 50, 754–763 (2018). https://doi.org/10.1038/s41588-018-0111-2

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