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scNanoHi-C: a single-cell long-read concatemer sequencing method to reveal high-order chromatin structures within individual cells

Nature Methods volume 20, pages 1493–1505 (2023)Cite this article

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Abstract

The high-order three-dimensional (3D) organization of regulatory genomic elements provides a topological basis for gene regulation, but it remains unclear how multiple regulatory elements across the mammalian genome interact within an individual cell. To address this, herein, we developed scNanoHi-C, which applies Nanopore long-read sequencing to explore genome-wide proximal high-order chromatin contacts within individual cells. We show that scNanoHi-C can reliably and effectively profile 3D chromatin structures and distinguish structure subtypes among individual cells. This method could also be used to detect genomic variations, including copy-number variations and structural variations, as well as to scaffold the de novo assembly of single-cell genomes. Notably, our results suggest that extensive high-order chromatin structures exist in active chromatin regions across the genome, and multiway interactions between enhancers and their target promoters were systematically identified within individual cells. Altogether, scNanoHi-C offers new opportunities to investigate high-order 3D genome structures at the single-cell level.

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Fig. 1: scNanoHi-C: a single-cell long-read method to detect proximal 3D chromatin contacts.
Fig. 2: Detecting chromatin structures and reconstruction of the haplotype-resolved 3D genome structure model in a single diploid cell using scNanoHi-C.
Fig. 3: Detection of cell-type-specific chromatin structures and genomic variations in single cells.
Fig. 4: Detecting high-order chromatin interactions in single cells.
Fig. 5: Identification of genomic region-specific synergies in GM12878 and COLO320DM cells.

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Data availability

The sequencing data of scNanoHi-C have been deposited in the Gene Expression Omnibus under accession code GSE217189. All other published datasets and annotation files used in this study are summarized in Supplementary Table 11. Source data files have been deposited in the figshare database at https://doi.org/10.6084/m9.figshare.23566704 (ref. 72). Source data are provided with this paper.

Code availability

Codes related to this paper can be accessed at https://github.com/LuJiansen/scNanoHi-C. Codes are also deposited into Zenodo (https://doi.org/10.5281/zenodo.7769419)73.

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Acknowledgements

We thank the Beijing Advanced Innovation Center for Genomics for support and part of the analysis was performed on the High-Performance Computing Platform of the Center for Life Sciences (Peking University). We thank the staff at the Peking University High-throughput Sequencing Center for help with flow cytometry. We thank D. Xing, Q. Xia, Y. Chen and Z. Liu (Peking University) for helpful suggestions for the Hi-C experiments. This work was supported by the Changping Laboratory, Beijing Advanced Innovation Center for Genomics at Peking University and the National Natural Science Foundation of China (32288102 to F.T.).

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  1. These authors contributed equally: Wen Li, Jiansen Lu.

Authors and Affiliations

  1. School of Life Sciences, Biomedical Pioneering Innovative Center, Peking University, Beijing, China

    Wen Li, Jiansen Lu, Ping Lu, Yun Gao, Yichen Bai, Kexuan Chen, Xinjie Su, Mengyao Li, Jun’e Liu, Lu Wen & Fuchou Tang

  2. Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Wen Li, Xinjie Su, Yijun Chen, Lu Wen & Fuchou Tang

  3. Beijing Advanced Innovation Center for Genomics (ICG), Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China

    Wen Li, Jiansen Lu, Ping Lu, Yun Gao, Kexuan Chen, Yijun Chen, Lu Wen & Fuchou Tang

  4. Changping Laboratory, Beijing, China

    Wen Li, Jiansen Lu & Fuchou Tang

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Contributions

F.T., W.L. and J. Lu conceived the project. W.L. was in charge of the experimental part and developed the scNanoHi-C protocol for the Nanopore platform with the help of J. Lu. Y.B. offered help for DNA FISH. J. Lu was in charge of the bioinformatics analysis with the help of W.L. and P.L. W.L., J. Lu and F.T. wrote the paper. Y.G., K.C., X.S., M.L., J. Liu, Y.C. and L.W. offered suggestions.

Corresponding author

Correspondence to Fuchou Tang.

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Competing interests

F.T., W.L. and J. Lu are investors on a patent that covers scNanoHi-C. The other authors declare no competing interests.

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Nature Methods thanks Bing Ren and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Lei Tang and Hui Hua, in collaboration with the Nature Methods team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 The detailed experimental process of scNanoHi-C.

a. Schematic diagram for detailed experimental procedure and principle of scNanoHi-C. b. The length distribution of fragments after MboI digestion (left), proximity ligation (medium) and amplification (right), respectively. c. Collisions rate of the whole experiment (left, n = 96) and permeabilized nuclei during amplification (right, n = 192). We identified collision cells as those in which less than 95% of monomers mapped to the mouse or to human genome (Supplementary Text 3).

Source data

Extended Data Fig. 2 The quality control of scNanoHi-C data.

a. An overview of data statistics of merged high-, medium-, and low-depth scNanoHi-C for GM12878. b. Composition of contacts of each single-cell from high-, medium- and low-depth data, respectively. Contacts were colored by their assigned groups during filtration, including adjacent, close, duplicate, promiscuous, isolated, and passed contacts. c. Composition of duplicate sets in each cell from high-, medium- and low-depth data, respectively. Duplicate sets were colored based on their group. ‘Identical’ means all concatemers are the same within the set, ‘included’ means all other contacts are included in the concatemer with the highest cardinality, and ‘others’ are the remaining duplicate sets. d. Boxplot showed the percentage of contacts within a duplicate set that were included in the concatemer with the highest cardinality, related to the ‘others’ group of (c). e-i. Total bases after removal of sequencing adaptors (e), ratio of passed contacts (f), ratio of trans-chromosomal contacts (g), the number of contacts (h), and ratio of high-order concatemers (i) in single-cell of high-, medium-, low-depth scNanoHi-C. j. The coverage of high-, medium- and low-depth scNanoHi-C in different resolutions (50 kb, 100 kb, 500 kb, and 1 Mb, respectively). The n = 24, 192 and 456 biologically independent cells for high-, medium- and low-depth scNanoHi-C, respectively (d-j). From left to right are the number of monomers (left) and contacts (middle) detected in each bin and the percentage of covered bins in each cell. The central lines of boxplots are median of data. The lower and upper hinges correspond to the 25th and 75th percentiles. The end of lower and upper whiskers are 1.5 * IQR (inter-quartile range). Data beyond the end of the whiskers are plotted as outliers (d-j).

Source data

Extended Data Fig. 3 Detecting 3D genome structures in merged scNanoHi-C.

a, b.(i) Correlation of 1-Mb raw contacts between merged high-, medium-depth scNanoHi-C and bulk Hi-C for GM12878 (r = 0.96, r = 0.96). (ii) Correlation of 1-Mb CS between merged high-, medium-depth scNanoHi-C and bulk Hi-C for GM12878 (r = 0.85, r = 0.93). (iii) Correlation of 50-kb IS between merged high-, medium-depth scNanoHi-C and bulk Hi-C for GM12878 (r = 0.78, r = 0.88). (iv) Comparison of aggregate peak analysis (APA) for merged high-, medium-depth scNanoHi-C virtual pair-wise contacts and bulk Hi-C pair-wise contact density within 100 kb of Hi-C loop anchors. c. Dependence of contact probability on genomic distance, P(s), for bulk Hi-C, low-, medium-, high-depth scNanoHi-C. d. Derivative of the P(s) plots from c. e. Dependence of normalized contact probability on genomic distance for bulk Hi-C, low-, medium-, high-depth scNanoHi-C.

Source data

Extended Data Fig. 4 Comparisons between scNanoHi-C and scSPRITE.

a. Distribution of cardinality at concatemer/cluster levels (left) and monomer/read levels (right) in scNanoHi-C (top) and scSPRITE (bottom), respectively. b. Distribution of cardinality at the cluster (left) and read (right) levels in single cells of scNanoHi-C (top) and scSPRITE (bottom). Cells were ordered by the number of contacts and clusters for scNanoHi-C and scSPRITE, respectively. Clusters or reads were grouped by their cardinality. c. The decay of normalized contacts probability as a function of genomic distance separation for single cells in scNanoHi-C and scSPRITE. Contacts from each cell were grouped by their cardinality, the lines and shadows represent the mean and 25% to 75% quantiles of normalized contacts probability among all cells, respectively. The black line represents the result of bulk in situ Hi-C. d. The ratio of trans-chromosomal contacts (trans-ratio) in single cells of scNanoHi-C and scSPRITE (left). The trans-ratios of scSPRITE were further calculated by each cardinality group (right). The n = 1,000, 288 and 288 biologically independent cells for scSPRITE, medium- and low-depth scNanoHi-C, respectively. The central lines of boxplots are the median of data. The lower and upper hinges correspond to the 25th and 75th percentiles. The end of the lower and upper whiskers are 1.5 * IQR (inter-quartile range). e. Aggregate peak analysis (APA) for mESC of bulk in situ HiC (top), scSPRITE (middle) and scNanoHi-C (bottom) within 100 kb on either side of loop anchors identified from bulk Hi-C. Contacts of scSPRITE and scNanoHi-C were merged according their cardinality. The number on the upper left corner represents the enrichment score of contacts in the central pixel.

Source data

Extended Data Fig. 5 Detecting 3D genome structures in single cells using scNanoHi-C data.

a–c. Distribution of average normalized detection scores (nDS) across all cells in each pairs of chromosome territories (n = 253, 1-Mb resolution, a), A/B compartment switching triplets (A-B-A or B-A-B, n = 630, 1-Mb resolution, b) and TAD boundaries (n = 2,503, 50-kb resolution, c) of GM12878 scNanoHi-C data; score = 0 (red line). Representative structures from a medium- and low-depth GM12878 scNanoHi-C cell were shown at the right. All chromosome territories (chr1 to chr22 and chrX) in a (ii) was visulazed at 5-Mb resolution. d. Percentage of cells with TAD nDS > 0 for each TAD boundary; e. Correlation between average TAD nDSs and TAD boundary strength of each TAD boundary. Spearman’s correlation was calculated and tested by ‘cor.test’ function in R with default two-sided test. f. comparison between the average TAD normalized detection scores in boundaries across 672 biologically independent cells with (n = 1,506) and without (n = 997) the presence of CTCF binding (inferred from CTCF ChIP-seq peaks). Paired two-side t-test was used to determine the significance. The central lines of boxplots are median of data. The lower and upper hinges correspond to the 25th and 75th percentiles. The end of lower and upper whiskers are 1.5 * IQR (inter-quartile range). Data beyond the end of the whiskers are plotted as outliers.

Source data

Extended Data Fig. 6 Identifying loops with scNanoHi-C.

a. Information about the datasets used for detecting loops with SnapHiC and the number of detected loops in each dataset. b. Track view of WashU epigenome browser showing two representative clusters of loops around MIR155HG and RUNX3. From top to bottom are refGene and repeatMasker annotations, ChIP-seq signals of CTCF, H3K4me3, H3K27ac, and RAD21, loops identified from scNanoHi-C, cohesin HChIP (called with FitHiChIP), cohesin HiChIP (called with MAPS), H3K27ac HiChIP (called with FitHiChIP), H3K27ac HiChIP (called with MAPS) and in situ Hi-C (called with HiCCUPS). Super-enhancers were highlighted with orange shadows. c. Track view of WashU epigenome browser showing two representative clusters of loops around Sox2 and Nanog. From top to bottom are refGene and repeatMasker annotations, ChIP-seq signals of H3K4me1, H3K4me3, and H3K27ac, loops identified from scNanoHi-C, cohesin HChIP (called with FitHiChIP) and in situ Hi-C (called with HiCCUPS).

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Extended Data Fig. 7 Reconstruction of the 3D genome structure model in single cells using scNanoHi-C data.

a. The distribution of median monomer length of merged high-, medium-, low-depth scNanoHi-C for GM12878 (left); the distribution of median concatemer length of merged high-, medium-, low-depth scNanoHi-C for GM12878 (right). b. The distribution of median concatemer length of a representative single cell (left); the distribution of median monomer length of a representative single cell (right). c. Semi-phased (left) and full-phased (right) percentage of monomers in Dip-C (n = 16) and low- (n = 456), medium- (n = 192), high-depth (n = 24) scNanoHi-C (left). The central lines of boxplots are median of data. The lower and upper hinges correspond to the 25th and 75th percentiles. The end of lower and upper whiskers are 1.5 * IQR (inter-quartile range). Data beyond the end of the whiskers are plotted as outliers. d. Rank-normalized 1-Mb 3D scA/B values of maternal (red dot) and paternal alleles (blue dot) in GM12878 at chr1 to chr22 and chrX (RMS r.m.s.d. < 1.5, n = 28). e. Radial position of maternal (red dot) and paternal alleles (blue dot) in GM12878, as measured by average distances to the nuclear center (RMS r.m.s.d. < 1.5, n = 28).

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Extended Data Fig. 8 Detecting CNVs in single cells using scNanoHi-C data.

a. CNVs of merged GM12878 cells at in 1-Mb resolution, copy numbers across the genome are plotted in merged GM12878 cells from high-, medium-, low-depth of scNanoHi-C. b. The relationship between numbers of contacts and CV at different resolutions of GM12878 scNanoHi-C data. c. The heat map of CNV profiles in GM12878 scNanoHi-C single cells without clear clonal CNVs (contacts > 100k, n = 169),1-Mb windows (left); CNV profiles of MALBAC GM12878 single cells (n = 147, number of reads > 1 million, CV < 0.25, left). Cells without and with CNVs (length > 20 Mb, supported by at least 5 cells) were selected and visualized at the top and bottom, respectively. d. The average distance of particles within paternal and maternal chr1 (left, n = 25,200 particles at 20 kb, P-value was determined by paired two-side t-test), the long-arm (n = 7,260) and short-arm (n = 5,151) of paternal (middle, P-value was determined by unpaired one-side t-test) and maternal (right, P-value was determined by unpaired one-side t-test) chr1 of the same cell mentioned in Fig. 3g. The central lines of boxplots are the median of data. The lower and upper hinges correspond to the 25th and 75th percentiles. The end of the lower and upper whiskers are 1.5 * IQR (inter-quartile range). e. The relationship between numbers of contacts and CV at different resolutions of K562 scNanoHi-C data. f. CNV profiles of K562 WGS, merged scNanoHi-C, bulk Hi-C and three representative single cells of scNanoHi-C at 1-Mb resolution.

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Extended Data Fig. 9 Analyses of the relationships between high-order ratios and other factors.

a, b. Heat map showed the partial Spearman’s correlation analyses between the ratio of high-order concatemers and other factors among each 50-kb genomic windows in GM12878 (a.) and K562 (b), respectively. c. Partial Pearson’s correlations between the conditioned ratio of high-order concatemers and the average length of monomers in K562. d. Partial Pearson’s correlations between the conditioned ratio of high-order concatemers and compartment scores in K562. e–f. the distribution of supported concatemers and cells in one enhancer bin regulating multiple promoter bins (e.) and one promoter bin regulated by multiple enhancer bins (f.).

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Extended Data Fig. 10 Detecting synergies in single GM12878 cells and single COLO320DM cells.

a. The LOLA enrichment of GM12878 regulatory elements synergies (n = 917) relative to backgrounds (n = 1481); RNAPII, RNA polymerase II. The values of each bar represent log2(odd ratios) of enrichment for each feature, error bars represent the 95% confidence intervals. See also in Methods. b. The DNA FISH of MYC in COLO320DM. The experiment were repeated two times independently with similar results in multiple views. c. The contacts map of COLO320DM in merged scNanoHi-C and single-cell scNanoHi-C data; red triangles, regions of MYC gene amplification. d. The CNVs pattern of single COLO320DM cells, 1-Mb resolution. e. The adjnTIF of merged single COLO320DM cells at genome-wide scale. f. Single−cell averaged adjnTIF of COLO320DM at genome-wide scale. g. AdjnTIF of ecDNA region in single COLO320DM cells. The central lines of boxplots are the median of data. The lower and upper hinges correspond to the 25th and 75th percentiles. The end of the lower and upper whiskers are 1.5 * IQR (inter-quartile range).

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Li, W., Lu, J., Lu, P. et al. scNanoHi-C: a single-cell long-read concatemer sequencing method to reveal high-order chromatin structures within individual cells. Nat Methods 20, 1493–1505 (2023). https://doi.org/10.1038/s41592-023-01978-w

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