DNA immunoprecipitation followed by sequencing (DIP-seq) is a common enrichment method for profiling DNA modifications in mammalian genomes. However, the results of independent DIP-seq studies often show considerable variation between profiles of the same genome and between profiles obtained by alternative methods. Here we show that these differences are primarily due to the intrinsic affinity of IgG for short unmodified DNA repeats. This pervasive experimental error accounts for 50–99% of regions identified as ‘enriched’ for DNA modifications in DIP-seq data. Correction of this error profoundly altered DNA-modification profiles for numerous cell types, including mouse embryonic stem cells, and subsequently revealed novel associations among DNA modifications, chromatin modifications and biological processes. We conclude that both matched input and IgG controls are essential in order for the results of DIP-based assays to be interpreted correctly, and that complementary, non-antibody-based techniques should be used to validate DIP-based findings to avoid further misinterpretation of genome-wide profiling data.

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This work was supported by the Swedish Research Council (2015-03495 to C.E.N.; 2015-02575 to M.B.), LiU-Cancer (2016-007 to C.E.N.), the Swedish Cancer Society (CAN 2017/625 to C.E.N.; CAN 2016/602 to H.G.) and the Medical Research Council, UK (MC_PC_U127574433 to R.R.M. and H.K.M.).

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Author notes

  1. These authors contributed equally: Mikael Benson and Colm E. Nestor.


  1. Division of Pediatrics, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

    • Antonio Lentini
    • , Cathrine Lagerwall
    • , Karolos Douvlataniotis
    • , Hartmut Vogt
    • , Mikael Benson
    •  & Colm E. Nestor
  2. Division of Drug Research, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden

    • Svante Vikingsson
    •  & Henrik Green
  3. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden

    • Svante Vikingsson
    •  & Henrik Green
  4. MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK

    • Heidi K. Mjoseng
    •  & Richard R. Meehan


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C.L., S.V., K.D. and H.K.M. performed experiments; A.L., C.E.N. and S.V. analyzed data; A.L., R.R.M. and C.E.N. wrote the manuscript; and H.V., H.G., R.R.M., M.B. and C.E.N. supervised the work.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Colm E. Nestor.

Integrated supplementary information

  1. Supplementary Figure 1 Reproducibility of off-target binding in DIP-seq between studies.

    Signal track for multiple marks and tissues in mice over repetitive regions. STRs, short tandem repeats. All tracks are automatically scaled.

  2. Supplementary Figure 2 Extended identification and validation of off-target binding in DIP-seq.

    (a, b) Immuno dot blot n = 1 (a) and ELISA n = 3 biologically independent experiments (b) of 5mC, 5hmC, 5fC and 5caC antibodies in synthetic 426 bp oligos containing the different marks. Boxplots represent median and first and third quartiles with whiskers extending 1.5 * inter-quartile range. (c) Enrichment of IgG or Input reads over the intersection of DIP-seq 5modC (5mC+5hmC+5fC+5caC) n = 592 enriched regions or non-intersecting (5mC/5hmC/5fC/5caC) n = 259002 enriched regions. Data represented as in b. P-values calculated using two-tailed T-test. (d) Correlation matrix of enriched DIP-seq regions per Mbp of mm9. Correlation was calculated as pairwise two-tailed Pearson correlation r2 for each n = 1 biologically independent experiment. (e) Venn diagram of overlapping enriched regions for 5hmC, 5mC and IgG (left). Dinucleotide frequencies for overlapping IgG+5mC+5hmC n = 23317 regions, 5mC+5hmC, n = 6683 regions and mm9 n = 23317 randomly sampled regions. Data represented as in b. (f) Number of methylated CpH from WGBS data per IgG n = 137557 enriched region or 5mC n = 19091 enriched region. P-values calculated using two-tailed Mann-Whitney U-test. (g) Enrichment profile of IgG and 5hmC in DnmtTKO (left) or TetTKO (right) and WT mESCs over IgG n = 137557 enriched regions. Data shown as mean for WT and DnmtTKO n = 1 biologically independent sample (left) and mean and 95% confidence intervals for WT n = 2 and TetTKO n = 3 biologically independent samples (right). (h) DIP using a 5hmC antibody in wild-type (WT) (left) and DnmtTKO (right) mESCs for DIP-qPCR n = 3 and DIP-seq n = 1 biologically independent samples. Data shown as mean ±s.d. Correlation between mean DIP-qPCR and DIP-seq values calculated using two-tailed Spearman correlation. (i) CG content of enriched fragments for DIP and Seal profiling for 5hmC (left) and 5fC (right). Theoretical normal distribution modelled based on mean and s.d. for each mark (Norm). P-values calculated by two-tailed Kolmogorov-Smirnov test using the mean of n = 2 biologically independent experiments. (j) Estimation of PCR duplication for sequencing libraries at a depth of 10 million reads shown as the non-redundant fraction (ie. not duplicated fraction) for n = 2 biologically independent samples. Data represented as in b.

  3. Supplementary Figure 3 Extended analysis of 6mA DIP-seq in multiple species.

    (a) Scatterplot showing correlation between IgG motif similarity for DIP-seq and percentage CA-repeats in the respective genomes for M. musculus n = 11, D. rerio n = 2, X. laevis n = 8, C. elegans n = 1 and E. coli n = 2 biologically independent samples. Correlation calculated as two-tailed Spearman's rho for all samples together (n = 24). Line represents linear correlation and 95% confidence interval. (b) Number and overlap of 6mA enriched regions in X. laevis testes identified using Input or IgG controls shown as Venn diagrams (left) and bar plots (right). (c) Number of reads mapping to Mycoplasma species. Kidney n = 3, mESC n = 2, Brain n = 6 biologically independent samples. Boxplots represent median and first and third quartiles with whiskers extending 1.5 * inter-quartile range.

  4. Supplementary Figure 4 Effect of IgG correction in DIP-seq data.

    (a) Schematic visualization of false positive rate for enriched regions. Briefly, false positive rate (FPR) was estimated based on the inverse fraction of regions identified by both Input and IgG versus total regions. (b) Estimated false positive rate of enriched regions using IgG or Input as control for Tdg knockdown mESCs for n = 2 biologically independent samples. Data shown as mean. (c) Estimated false positive rate for individual mESC or MEF datasets. *Estimated based on controls from mESCs. (d) Venn diagram of enriched 5hmC regions in mESCs with different techniques and controls of each n = 1 biologically independent samples. (e, f) Fraction of enriched 5modC regions identified using IgG or Input overlapping repetitive elements (e) and dinucleotide repeats (f) for 5caC n = 2, 5fC n = 2, 5hmC n = 7 and 5mC n = 6 biologically independent samples. Presented as mean ± s.d. P-values calculated using two-tailed T-test. (g) Venn diagram of 5mC and 5hmC overlap using IgG or Input controls (top) and paired line plot of 5mC and 5hmC overlap using IgG or Input controls for multiple studies (indicated by symbols, bottom). Data shown as mean and individual data points of n = 6 biologically independent samples. P-values calculated using two-tailed paired T-test. ▲ = ERP000570, = GSE31343, ■ = GSE24841, = GSE42250. (h) GO term enrichment for top genes (n = 500) enriched for 5hmC in mouse embryonic fibroblasts (MEFs) using DIP-seq with either IgG or Input controls. P-values calculated using PANTHER overrepresentation test GO biological processes. (i) Signal track in mESCs of ChIP-seq controls over IgG DIP-seq enriched regions.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–4, Supplementary Discussion and Supplementary Table 5

  2. Reporting Summary

  3. Supplementary Table 1

    Summary of analyzed datasets and their relationship to figures

  4. Supplementary Table 2

    Motif-enrichment analysis of 21 published 5modC DIP-seq datasets

  5. Supplementary Table 3

    Motif-enrichment analysis of 23 published 6mA DIP-seq datasets

  6. Supplementary Table 4

    Analysis of cell-culture contamination in 36 DIP-seq datasets

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