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Methylation signatures in cell-free DNA (cfDNA) have shown great sensitivity and specificity in the characterization of tumour status and classification of tumour types, as well as the response to therapy and recurrence. Currently, most cfDNA methylation studies are based on bisulphite conversion, especially targeted bisulphite sequencing, while enrichment-based methods such as cfMeDIP-seq are beginning to show potential. Here, we report an enrichment-based ultra-low input cfDNA methylation profiling method using methyl-CpG binding proteins capture, termed cfMBD-seq. We optimized the conditions for cfMBD capture by adjusting the amount of MethylCap protein along with using methylated filler DNA. Our data show high correlation between low input cfMBD-seq and standard MBD-seq (&gt;1000 ng input). When compared to cfMEDIP-seq, cfMBD-seq demonstrates higher sequencing data quality with more sequenced reads passed filter and less duplicate rate. cfMBD-seq also outperforms cfMeDIP-seq in the enrichment of CpG islands. This new bisulphite-free ultra-low input methylation profiling technology has great potential in non-invasive and cost-effective cancer detection and classification.

Cell-free DNA methylome profiling by MBD-seq with ultra-low input