Abstract 2097: Cell-free DNA methylome profiling by MBD-seq with ultra-low input
Authors: Huang Jinyong , Soupir Alex C. , Wang Liang 
Published: July 2021

Methylation signatures in cell-free DNA (cfDNA) have shown great sensitivity and specificity in the characterization of tumor status and classification of tumor types, as well as the response to therapy and recurrence. Currently, most cfDNA methylation studies are based on bisulfite conversion. However, bisulfite treatment may result in the substantial degradation of up to 96% of DNA. This reduction critically impacts the assay's sensitivity, especially for low yield material such as cfDNA. Recently, enrichment-based cfMeDIP-seq that uses cfDNA as starting material is beginning to show potential. Here, we report another enrichment-based ultra-low input cfDNA methylation profiling method using methyl-CpG binding proteins capture, termed cfMBD-seq. We optimized the conditions of cfMBD capture by adjusting the amount of MethylCap protein and beads along with using exogenous λ DNA as filler DNA. Our data showed that cfMBD-seq performed equally to the standard MBD-seq (1000 ng input) even when using 1 ng DNA as the input. cfMBD-seq performed better than a previously published low input MBD-seq protocol without using filler DNA in the enrichment of methylated DNA, with 59.63% vs. 19.77% reads mapped to CpG islands and 94.74% vs. 85.66% reads mapped to combined CpG islands/shores/shelves. cfMBD-seq also demonstrated higher sequencing data quality when compared to cfMeDIP-seq, with more reads that passed filter and less duplicate rate (79.80% vs. 60.91% sequenced reads used for analyses). We showed that cfMBD-seq outperformed cfMeDIP-seq in the enrichment of fragments with higher CpG density (29.59 vs. 23.33, number of CpG per fragments where coverage reaches peak), CpG islands for example (59.63% vs. 38.89% reads mapped on CpG islands). cfMBD-seq also successfully recapitulates methylation profiles from reduced representation bisulfite sequencing and whole-genome bisulfite sequencing. Overall, cfMBD-seq is a method of choice for interrogating epigenetic regulation of gene expression (methylation changes on CpG islands). On the other hand, cfMeDIP-seq would be preferable in investigating transcriptional regulation of non-coding RNAs (methylation changes on gene bodies and CpG shores). Our study demonstrates the potential benefits of using cfMBD-seq to profile the methylome of cfDNA with ultra-low DNA input. Current results provide justification for further validation using case and control plasma samples from different malignancies to perform differential methylation analyses. Another potential for cfMBD-seq is the use in other methylome-wide investigations that are limited by DNA yield. This new bisulfite-free ultra-low input methylation profiling technology has a great potential in non-invasive and cost-effective cancer detection and classification.

Citation Format: Jinyong Huang, Alex C. Soupir, Liang Wang. Cell-free DNA methylome profiling by MBD-seq with ultra-low input [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2097.