Abstract
Cell-free DNA (cfDNA) in human plasma provides access to molecular information about the pathological processes in the organs or tumors from which it originates. These DNA fragments are derived from fragmented chromatin in dying cells and retain some of the cell-of-origin histone modifications. In this study, we applied chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 268 human samples. In healthy donors, we identified bone marrow megakaryocytes, but not erythroblasts, as major contributors to the cfDNA pool. In patients with a range of liver diseases, we showed that we can identify pathology-related changes in hepatocyte transcriptional programs. In patients with metastatic colorectal carcinoma, we detected clinically relevant and patient-specific information, including transcriptionally active human epidermal growth factor receptor 2 (HER2) amplifications. Altogether, cfChIP-seq, using low sequencing depth, provides systemic and genome-wide information and can inform diagnosis and facilitate interrogation of physiological and pathological processes using blood samples.
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Data availability
Data collected in this study were deposited in the European Genome-phenome Archive (EMBL-EBI) repository with accession code EGAS00001004913. BED files and browser tracks are available in the Zenodo repository: https://doi.org/10.5281/zenodo.3967253.
Browser tracks can be viewed by the UCSC genome browser.
• Session: http://genome.ucsc.edu/s/nirfriedman/cfChIP-seq
• Track hub: http://www.cs.huji.ac.il/~nir/Hubs/cfChIP-seq/hub.txt
Additional data from public repositories are listed here:
The datasets are as follows: UCSC known genes (AH5036); Ensembl transcripts (AH5046); genomic annotations (AH5040): AnnotationHub (http://bioconductor.org/packages/release/bioc/html/AnnotationHub.html); consolidated ChIP-seq: Roadmap Epigenomics (https://egg2.wustl.edu/roadmap/data/byFileType/alignments/consolidated/); mRNA-seq: Roadmap Epigenomics (https://egg2.wustl.edu/roadmap/data/byDataType/rna/expression/57epigenomes.RPKM.pc.gz); consolidated ChromHMM calls: Roadmap Epigenomics (http://egg2.wustl.edu/roadmap/data/byFileType/chromhmmSegmentations/ChmmModels/coreMarks/jointModel/final/all.mnemonics.bedFiles.tgz).
Code availability
R code for processing cfChIP-seq data is available at https://github.com/nirfriedman/cfChIP-seq.git.
Change history
01 March 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41587-021-00853-3
21 January 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41587-021-00831-9
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Acknowledgements
We thank N. Kaminski, J. Moss, E. Pikarsky, N. Rajewsky, O.J. Rando, A. Regev and members of the Friedman lab for discussions and comments on this manuscript. We thank L. Friedman for help with illustrations and graphics. This work was supported by the European Research Council’s AdG Grants 340712 ‘ChromatinSys’ (to N.F.) and 786575 ‘RxmiRcanceR’ (to E.G.); the Israel Science Foundation’s I-CORE program grant 1796/12 (to T.K. and N.F.) and grants 2612/18 (to N.F.), 3020/20 (to A.G.), 2473/17 (to E.G.) and 486/17 (to E.G.); Israel Ministry of Science and Technology grant 3-14352 (to A.G.); National Institutes of Health grants RM1HG006193 (to N.F.) and CA197081-02 (to E.G.); Deutsche Forschungsgemeinschaft SFB841 (to E.G.); and DKFZ-MOST grant (to E.G.).
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Contributions
R. Sadeh and N.F. developed the concept. R. Sadeh, N.F. and I.S. designed the experiments with help from E.G., B.G., A.Z. and Y.D. R. Sadeh developed the cfChIP-seq method with help from A.R. and I.S. I.S., R. Sadeh and A.R. performed cfChIP-seq experiments. N.F. and G.F. developed analytical tools with help from J.G., M.N., G.M. and T.K. N.F., R. Sadeh, G.F., I.S. and J.G. analyzed the data. I.F.F, D.N. and R. Shemer performed the cfDNA methylation assays. Z.K. collected healthy donor samples. D.Y., T.P., A.H., J.E.C., A.S., M.T., A.G., M.M., S.A.G., A.B.Y., E.S., R. Safadi, D.P., E.G., B.G. and A.Z. provided clinical insights, recruited patients and collected patient samples. N.F., R. Sadeh, J.G., G.F. and A.C. wrote the paper with input from all authors.
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A patent application for cfChIP-seq has been submitted by the Hebrew University of Jerusalem. R. Sadeh, I.S., J.G. and N.F. are founders of Senseera.
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Extended data
Extended Data Fig. 1 Supporting data for Fig. 1.
a, Distribution of reads for cfChIP-seq with different antibodies on four samples (H012.1, H012.2, H013.1, and H013.2). We divided the genome into regions that contain (putative) TSS based on our catalogue (see below) and (putative) Enhancers. Since there are regions that are marked as both (in different tissues), we consider the intersection separately. For each subset we show the fraction of reads mapped to the region. Within each bar, the fraction estimated as background (based on our background model, Methods) is marked in dark gray. b, Genome browser view (as in Fig. 1c). c, Metaplots (as in Fig. 1d) of ChIP-seq samples from the Roadmap Epigenomics compendium. d, Scatter plots showing signal levels from cfChIP-seq versus Leukocyte ChIP-seq of H3K4me3, H3K4me2, and H3K36me3 (similar to Fig. 1e). e, Estimation of the amount of specific reads in cfChIP-seq. Top panel: box plot of the estimate of % reads that are above background levels for all the cfChIP-seq samples analyzed in the manuscript (Supplementary Table 1) compared to selected ChIP-seq samples from Roadmap Epigenomics compendium. Bottom panel: percent of the signal above background that is in the expected genomic locations (i.e H3K4me1 and H3K4me2 - promoters and enhancers, H3K4me3 - promoters, H3K36me3 - gene bodies). For comparison, the same analysis pipeline was applied to selected Roadmap Epigenomic ChIP-seq samples against the same marks. Box limits: 25% –75% quantiles, middle: median, upper (lower) whisker to the largest (smallest) value no further than 1.5 * inter-quartile range from the hinge.
Extended Data Fig. 2 Supporting data for Fig. 1.
a, Fragment length distribution for all samples in this manuscript. Each row represents a histogram of fragment length of a specific sample. Color represents the number of fragments/million with that length (RPM). b, Reproducibility of the cfChIP-seq assay. Shown are technical repeats, biological repeats (two samples from the same donor) and comparison of two different donors for three histone marks. Each dot is a gene, and values are normalized counts at the gene promoter (H3K4me2/3) or body (H3K36me3).
Extended Data Fig. 3 Supporting data for Fig. 2.
a, Testing gene sets defined by highly expressed in different cancer types (TCGA, Methods) against genes with higher signal in a CRC tumor sample (Fig. 2a). Hypergeometric test with FDR corrected q-values. b, Levels of H3K4me2 coverage over colon-specific enhancers (y-axis) in healthy donors and in CRC cancer samples. Box limits: 25% –75% quantiles, middle: median, upper (lower) whisker to the largest (smallest) value no further than 1.5 * inter-quartile range from the hinge, n = 144. c, Average coverage of H3K36me3 across gene bodies (meta gene). d, Coverage of H3K36me3 cfChIP-seq over gene bodies in a healthy donor (H012.1) for genes at different leukocyte expression quantiles. Box limits: 25% –75% quantiles, middle: median, upper (lower) whisker to the largest (smallest) value no further than 1.5 * inter-quartile range from the hinge.
Extended Data Fig. 4 Supporting data for Fig. 3.
a, Comparison of H3K4me3 cfChIP-seq signal from a healthy donor (H012.1) with expected gene expression levels, based on the expression in cells contributing to cfDNA in healthy subjects (Methods). Each dot is a gene. x-axis: normalized number of H3K4me3 reads in gene promoter. y-axis: expected expression in number of transcripts/million (TPM). b, Comparison (as in A) of Leukocytes H3K4me3 ChIP-seq signal vs. Leukocytes gene expression levels (both for Roadmap Epigenomic sample E062). c, Comparison (as in A) of H3K4me3 cfChIP-seq signal from a healthy donor (H012.1) vs. Liver gene expression levels (Roadmap Epigenomics sample E066). d, Summary of correlations of healthy cfChIP-seq levels against different expression patterns from Roadmap Epigenomics and BLUEPRINT. For each category of expression profiles we plot the boxplot of r2 values. Red line denotes the correlation against the predicted expression mixture of cells contributing to cfDNA pool (panel A). Box limits: 25% –75% quantiles, middle: median, upper (lower) whisker to the largest (smallest) value no further than 1.5 * inter-quartile range from the hinge. e, Comparison of the expression levels of genes in two clusters of Fig. 3c (see inset). Cluster A contains 4,690 genes that change between samples, and Cluster B contains 10,177 genes that do not change between samples. Violin plots show the distribution of expression levels in three tissues - PBMC, Heart, and Liver, from the Roadmap Epigenomics expression data. f, Overlap of both clusters with the set of genes with CpG island promoters (blue) and housekeeping genes (green; based on analysis of GTEX compendium, see Methods). For clarity we show each cluster in a separate Venn diagram.
Extended Data Fig. 5 cfChIP-seq is highly sensitive.
a, Schematics of the parameters involved in determining cfChIP-seq sensitivity. 1. Number of informative nucleosomes is the total number of signature-specific nucleosomes in the plasma that carry a mark of interest; 2. The percent contribution of the signature-positive cells to the circulation; 3. Total number of genomes in circulation; 4. The specific capture probability of marked nucleosomes by the cfChIP-seq assay; and 5. The non-specific capture probability of nucleosomes (background). The signal to noise ratio (SNR) is the ratio of the specific to non-specific capture probabilities. b, Simulation analysis of event detection power as a function of percent positive (x-axis) and number of informative locations (y-axis). Detection is defined as 95% probability of assay results (capture & sequencing) that reject the null hypothesis of background signal with p < 0.05 (Poisson test, Methods). Simulation assumes number of genomes = 10,000 (10 ml plasma of healthy donor), capture probability of 1%, and SNR of 500 (Methods, Supplementary Note). The size of several example signatures are shown.
Extended Data Fig. 6 Sensitivity analysis.
a, Total sizes (in nucleosomes) of TSS (Left) and Enhancer (Right) signatures of various cell types. b, Estimates of specific capture rate and of SNR (specific capture / non-specific capture) over 88 healthy samples, assuming 1000 genomes/ml and 2 ml input. Box limits: 25% –75% quantiles, middle: median, upper (lower) whisker to the largest (smallest) value no further than 1.5 * inter-quartile range from the hinge. c, Signal level is linear with input. Plasma of a healthy donor was spiked in with different amounts of yeast nucleosomes (x-axis). The number of counts observed (y-axis) for signatures of different sizes. Error bars show 20-80% range over 100 different sampled signatures of the given size. d, Genome browser of chrY male-specific promoters (left) and a representative autosomal region (right) in the male/female titration experiment. e, Test of sensitivity using male spike-in. Plasma of healthy female and male donors were titrated at different ratios. Detection of male-specific promoters as a function of percent of chrY genomes in the sample (x-axis). Shown are the number of counts (y-axis) and significance (circle radius) of signal above background distribution (Methods). f, Simulation study of the effect of capture probability on detection. The blue marks denote the concentrations used in the male-female titration experiment which had capture probabilities ~0.1% and SNRs of ~500-800. g, Simulation study of the effect of SNR levels on detection probability.
Extended Data Fig. 7 cfChIP-seq liver signal is proportional to % liver cfDNA.
a, % Liver as estimated using DNA CpG methylation markers vs. signature strength. b, % Liver as estimated using DNA CpG methylation markers vs. estimate of % liver in Fig. 5a.
Extended Data Fig. 9 Supporting data for Fig. 6.
a, Levels of CRC associated genes in different samples. Each point is a sample plotted with % CRC (x-axis) vs. normalized number of reads of the gene (y-axis). Solid points - the signal of the gene is significantly above the expectation given % CRC (Methods). b, Example of immune-related genes in CRC samples. Same as (A). c, Clustering of gene set enrichment in CRC samples (see Supplementary Table 11). d, Venn diagram of overlaps between cancer gene signatures that were identified in our analysis. e, Evaluation of cancer signatures in CRC samples from TCGA, grouped by their CMS subtype. Box limits: 25% –75% quantiles, middle: median, upper (lower) whisker to the largest (smallest) value no further than 1.5 * inter-quartile range from the hinge.
Supplementary information
Supplementary Information
Supplementary Note.
Supplementary Table 1
Sequencing statistics for samples sequenced in this study
Supplementary Table 2
Genes with abnormal signal (per sample)
Supplementary Table 3
Tumor signatures
Supplementary Table 4
Individuals and samples clinical information
Supplementary Table 5
Cell type signatures
Supplementary Table 6
Full analysis of tissue signatures versus samples
Supplementary Table 7
Full analysis of gene sets versus samples with reference
Supplementary Table 8
Differentially marked genes in pairwise comparisons
Supplementary Table 9
Liver clusters enrichments
Supplementary Table 10
Gene set counts in CRC samples relative to healthy reference
Supplementary Table 11
CRC signature enrichments
Supplementary Table 12
Roadmap Epigenomics samples used
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Sadeh, R., Sharkia, I., Fialkoff, G. et al. ChIP-seq of plasma cell-free nucleosomes identifies gene expression programs of the cells of origin. Nat Biotechnol 39, 586–598 (2021). https://doi.org/10.1038/s41587-020-00775-6
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DOI: https://doi.org/10.1038/s41587-020-00775-6
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