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

Abstract

Summary: 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. Motivation: For robust quantitative comparisons between samples, immunoprecipitation enrichment methods such as cfMeDIP-seq require normalization against common reference controls. Common reference controls can correct for technical variation in the processing of different samples. They can also correct for biases in enrichment due to known biophysical properties of DNA fragments such as fragment length, G + C content, and fraction of CpG dinucleotides. Furthermore, common reference controls can provide an experimental standard for quality control. We sought to provide synthetic spike-in DNA fragments that would fulfill these reference control purposes in cfMeDIP-seq experiments.

Keywords

• cell-free methylated DNA immunoprecipitation
• cfMeDIP
• spike-in controls
• DNA methylation
• cell-free DNA
• cfDNA