Sensitive and reproducible cell-free methylome quantification with synthetic spike-in controls

Cell Rep Methods. 2022 Sep 9;2(9):100294. doi: 10.1016/j.crmeth.2022.100294. eCollection 2022 Sep 19.


Cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) identifies genomic regions with DNA methylation, using a protocol adapted to work with low-input DNA samples and with cell-free DNA (cfDNA). We developed a set of synthetic spike-in DNA controls for cfMeDIP-seq to provide a simple and inexpensive reference for quantitative normalization. We designed 54 DNA fragments with combinations of methylation status (methylated and unmethylated), fragment length (80 bp, 160 bp, 320 bp), G + C content (35%, 50%, 65%), and fraction of CpG dinucleotides within the fragment (1/80 bp, 1/40 bp, 1/20 bp). Using 0.01 ng of spike-in controls enables training a generalized linear model that absolutely quantifies methylated cfDNA in MeDIP-seq experiments. It mitigates batch effects and corrects for biases in enrichment due to known biophysical properties of DNA fragments and other technical biases.

Keywords: DNA methylation; absolute quantification; batch effects; cell-free DNA; cell-free methylated DNA immunoprecipitation; cfDNA; cfMeDIP; early detection of cancer; liquid biopsy; minimally invasive testing; reference standards; spike-in controls.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Free Nucleic Acids* / genetics
  • DNA / genetics
  • DNA Methylation
  • Epigenome*
  • Genomics / methods


  • DNA
  • Cell-Free Nucleic Acids

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