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Comparison and imputation-aided integration of five commercial platforms for targeted DNA methylome analysis


Targeted bisulfite sequencing (TBS) has become the method of choice for the cost-effective, targeted analysis of the human methylome at base-pair resolution. In this study, we benchmarked five commercially available TBS platforms—three hybridization capture-based (Agilent, Roche and Illumina) and two reduced-representation-based (Diagenode and NuGen)—across 11 samples. Two samples were also compared with whole-genome DNA methylation sequencing with the Illumina and Oxford Nanopore platforms. We assessed workflow complexity, on/off-target performance, coverage, accuracy and reproducibility. Although all platforms produced robust and reproducible data, major differences in the number and identity of the CpG sites covered make it difficult to compare datasets generated on different platforms. To overcome this limitation, we applied imputation and show that it improves interoperability from an average of 10.35% (0.8 million) to 97% (7.6 million) common CpG sites. Our study provides guidance on which TBS platform to use for different methylome features and offers an imputation-based harmonization solution that allows comparative, integrative analysis.

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Fig. 1: Technology and design comparison of TBS platforms.
Fig. 2: Sequencing performance by the platform.
Fig. 3: Platform similarity and feature annotation.
Fig. 4: Platform reproducibility and concordance of DNA methylation calls.
Fig. 5: Differential methylation calls by the platform and imputation.

Data availability

The datasets generated and analyzed in the current study, including all raw targeted bisulfite sequencing, WGBS of Ref.gDNA and Nanopore sequencing data, have been deposited in the European Nucleotide Archive repository under accession number PRJEB46506 and are freely available. Raw WGBS sequencing data for the Coriell-NA12878 WGBS_EC sample generated by the ENCODE Project Consortium26 were downloaded from the ENCODE Project (experiment: ENCSR890UQO, library: ENCLB898WPW) (, and CpG count files for WGBS_IL sample were downloaded from Illumina BaseSpace Hub ( under sample name WGBS_P3 from HiSeq 4000: TruSeq DNA Methylation (NA12878, 2 × 76) dataset.

Code availability

The code used for annotation, differential methylation analysis, plotting and imputation is available in the GitHub repository at


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M.T. received funding from the European Union’s Seventh Framework Programme (Marie Skłodowska-Curie Actions FP7/2007-2013/WHRI-ACADEMY-608765); the Danish Council for Strategic Research (1309-00006B); the Ministry of Education, Science and Technological Development of Serbia (2011-2019/III-41026 and 451-03-68/2020-14/200043); and the Science Fund of the Republic of Serbia (PROMIS/2020/6060876). I.M. is supported by the Biotechnology and Biological Sciences Research Council (grant no. BB/M009513/1). S.B. has received funding from the Wellcome Trust (218274/Z/19/Z) and a Royal Society Wolfson Research Merit Award (WM100023). A.F. received support from the UCL/UCLH Biomedical Research Centre, the Medical Research Council (MR/M025411/1), Prostate Cancer UK (MA_TR15_009) and the Biotechnology and Biological Sciences Research Council (BB/R009295/1). S.R. received funding from Orchid. We further acknowledge support from D. Turner and B. Sipos (Oxford Nanopore Technologies) for the generation of the Nanopore sequencing data and from the CRUK–UCL Centre-funded Genomics and Genome Engineering and Bioinformatics Translational Technology Platforms.

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Authors and Affiliations



M.T., A.F. and S.B. conceived and designed the study. M.T. and S.R. performed the hybridization capture and RRBS experiments. P.D. and H.V. sequenced the libraries. M.T. and J.B. processed raw sequencing data. M.T. performed analysis of TBS data. I.M. analyzed WGBS and Nanopore data and performed imputation analysis. M.T., A.F. and S.B. interpreted the results. M.T., A.F. and S.B. wrote the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Miljana Tanić or Stephan Beck.

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Competing interests

The authors declare no competing interests.

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Nature Biotechnology thanks Miguel Branco, Alexander Dobrovic and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Sequencing data processing quality metrics produced by MultiQC.

a Bismark alignment rates for uniquely, ambiguously, or unaligned reads for each sample by platform; b Percent of reads aligning to top or bottom DNA strand for each sample by the platform; c Global methylation levels of CpG dinucleotides for each sample by the platform; d The global cytosine methylation level in CHG context for each sample by the platform used an estimate of sodium bisulfite under-conversion rates; e The global cytosine methylation level in CHH context for each sample by the platform used an estimate of sodium bisulfite under-conversion rates.; f -g M-bias plot shows the average percentage methylation and coverage across read length for each sample. Each line represents a sample. Methylation bias for the forward sequencing read by platform (f); Methylation bias for the reverse sequencing read by platform (g).

Extended Data Fig. 2 Target depth of coverage.

The fraction of targets covered at specific depth of sequencing for each sample by the platform: Agilent (a), Illumina (b) and Roche (c). Each sample is represented by a line.

Extended Data Fig. 3 Intra-platform concordance.

Scatterplot showing pairwise Pearson correlation coefficient for Coriell NA12878 data from, WGBS EC vs. WGBS IL (a), Nanopore vs. WGBS IL (b), and Nanopore vs. WGBS EC (c).

Extended Data Fig. 4 Platform interoperability.

Interoperability between platforms for Coriell NA12878 (left) and Ref.gDNA (right) before imputation (first row), after imputation without distnce treshold (sencond row), after imputation with 1000 bp distance treshold (third row) and after imputauion with 25 bp distance treshold (fourth row). Venn diagram showing CpGs overlapping between the platforms.

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Tanić, M., Moghul, I., Rodney, S. et al. Comparison and imputation-aided integration of five commercial platforms for targeted DNA methylome analysis. Nat Biotechnol 40, 1478–1487 (2022).

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