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Identifying high-confidence capture Hi-C interactions using CHiCANE

Abstract

The ability to identify regulatory interactions that mediate gene expression changes through distal elements, such as risk loci, is transforming our understanding of how genomes are spatially organized and regulated. Capture Hi-C (CHi-C) is a powerful tool to delineate such regulatory interactions. However, primary analysis and downstream interpretation of CHi-C profiles remains challenging and relies on disparate tools with ad-hoc input/output formats and specific assumptions for statistical modeling. Here we present a data processing and interaction calling toolkit (CHiCANE), specialized for the analysis and meaningful interpretation of CHi-C assays. In this protocol, we demonstrate applications of CHiCANE to region capture Hi-C (rCHi-C) and promoter capture Hi-C (pCHi-C) libraries, followed by quality assessment of interaction peaks, as well as downstream analysis specific to rCHi-C and pCHi-C to aid functional interpretation. For a typical rCHi-C/pCHi-C dataset this protocol takes up to 3 d for users with a moderate understanding of R programming and statistical concepts, although this is dependent on dataset size and compute power available. CHiCANE is freely available at https://cran.r-project.org/web/packages/chicane.

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Fig. 1: CHiCANE protocol workflow.
Fig. 2: Model fitting of count data.
Fig. 3: Interpretation of interaction peaks by distance.
Fig. 4: Replicate concordance by distance bins.
Fig. 5: eQTL analysis.
Fig. 6: Visualizing interaction peaks.
Fig. 7: Enrichment of enhancer marks.
Fig. 8: MK locus plot with TADs.
Fig. 9: Testing different models.

Data availability

Baitmaps for both sets of CHi-C libraries used in this study (rCHi-C T-47D10, pCHi-C MK9), hg38 HindIII in silico digest, HiCUP reports, CHiCANE’s unfiltered interactions, filtered interaction peaks (q-value < 0.05), and negative binomial model fit plots and statistics are available at https://doi.org/10.5281/zenodo.4073433.

Code availability

The CHiCANE R package is freely available through CRAN: https://cran.r-project.org/web/packages/chicane.

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Acknowledgements

We thank Breast Cancer Now for funding this work as part of Programme Funding to The Breast Cancer Now Toby Robins Research Centre. This study makes use of data generated by the PCHI-C Consortium. A full list of the investigators who contributed to the generation of the data is available in Javierre et al.9, which was funded by the National Institute for Health Research of England, UK Medical Research Council (MR/L007150/1) and UK Biotechnology and Biological Research Council (BB/J004480/1). We also thank D. Li from the WashU Epigenome Browser team for implementing support for CHiCANE’s standard format in the Epigenome Browser.

Author information

Affiliations

Authors

Contributions

E.M.H., O.F. and S.H. designed the study. E.M.H., O.C.L., O.F., F.D. and S.H. designed CHiCANE. E.M.H. and S.H. implemented the R package. E.M.H., A.G., O.C.L., G.M., O.S., O.F., F.D. and S.H. performed statistical experiments and interpreted data. J.B., A.Z., N.J., N.D. and L.B. generated capture Hi-C data. E.M.H., A.G., O.F. and S.H. wrote the manuscript with contributions from all authors. Y.C. and I.K. implemented dissemination of processed data. O.F. and S.H. supervised the experiments.

Corresponding authors

Correspondence to Olivia Fletcher or Syed Haider.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Peter Robinson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Baxter, J. S. et al. Nat. Commun. 9, 1028 (2018): https://doi.org/10.1038/s41467-018-03411-9

Dryden, N. H. et al. Genome Res. 24, 1854–1868 (2014): https://doi.org/10.1101/gr.175034.114

Extended data

Extended Data Fig. 1 Visualizing interaction peaks in WashU Epigenome Browser.

Image from WashU Epigenome Browser showing non-bait to bait interaction peaks (q-value < 0.05) called at known breast cancer risk loci by CHiCANE in the Baxter T-47D libraries at a, 16q12.2 locus and b, 14q24.1 locus. Yellow boxes show the captured regions.

Extended Data Fig. 2 Model fitting of counts data (pCHi-C library).

Representative examples of hanging rootograms depicting the negative binomial model fits on the Javierre MK library. Observed counts are shown as histogram bins (gray bars) while the CHiCANE fitted expected counts distribution is in red. The y-axis represents square root transformed density estimates of observed (gray bars) and expected (red line) counts. For observed counts, the height of the bars is shifted to align the top of the bar with the expected counts fit. Bars above and below the reference line (x-axis) indicate over- and under-prediction by the CHiCANE model, respectively.

Extended Data Fig. 3 Interpretation of interaction peaks by distance.

Examples of interpretation of interaction calling on Javierre MK library. a, Bar plots showing the proportion of interaction peaks (q-value < 0.05) by type (cis interactions include bait-to-bait interactions). b, Bar plots showing the number of interaction peaks (q-value < 0.05) across distance bins. c, Bar plots showing breakdown of region 1–10 Mb shown in (b).

Supplementary information

Supplementary Table 1

Somatic mutations overlapping with 2q35 target fragments. Example of interaction peaks called by CHiCANE from 2q35 locus of T-47D library annotated (target fragments only) with PCAWG SNV/MNV data using bedtools intersect. Column vcf_info contains information about the variant including allelic fraction, number of reads supporting variant and reference alleles (in tumor sample) and variant’s classification. The column ‘vcf_file’ contains the name of the vcf file i.e a unique patient id recorded in the PCAWG study.

Supplementary Table 2

INDELs overlapping with 2q35 target fragments. Example of interaction peaks called by CHiCANE from the 2q35 locus of the T-47D library annotated (target fragments only) with PCAWG INDELs data using bedtools intersect. The column ‘vcf_info’ contains information about the variant including allelic fraction, number of reads supporting variant and reference alleles (in tumor sample) and variant’s classification. The column ‘vcf_file’ contains the name of the vcf file i.e a unique patient id recorded in the PCAWG study.

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Holgersen, E.M., Gillespie, A., Leavy, O.C. et al. Identifying high-confidence capture Hi-C interactions using CHiCANE. Nat Protoc 16, 2257–2285 (2021). https://doi.org/10.1038/s41596-021-00498-1

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