97P - GENIE-seq: A novel methylation sequencing method for effective and accurate identification of methylation markers from cfDNA

Background

DNA methylation sequencing holds promise for early cancer detection. However, conventional bisulfite conversion-based methods such as Accel-NGS Methyl-Seq are inadequate for cfDNA (cell-free DNA) methylation analysis due to cumbersome operation and exacerbating cfDNA degradation. We developed a novel methylation sequencing method GENIE-seq, aimed at achieving accurate epigenetic profiling of cfDNA.

Methods

We compared the analytical performance of GENIE-seq, a high-fidelity method based on gentle enzymatic conversion with minimal DNA damage and convenient “one-tube” workflow, with a state-of-the-art method Accel-NGS Methyl-Seq. The library complexity, assay sensitivity, and methylation accuracy of both methods were analyzed using gDNA from HCT116 and GM12878 cell lines, and cfDNA from healthy donors. Accuracy of methylation level (β value) quantification was assessed using human gDNA reference materials with certain methylation levels. The robustness of GENIE-seq and Accel-NGS Methyl-Seq was evaluated with varying input amounts of cfDNA, and the impact of potential interferents was assessed.

Results

In comparing GENIE-seq libraries to Accel-NGS Methyl-Seq, it was found that GENIE-seq exhibited about 80% higher unique molecules regardless of sequencing depth. Furthermore, GENIE-seq demonstrated greater power in mutation detection ability compared to Accel-NGS Methyl-Seq. Additionally, GENIE-seq displayed superior accuracy in methylation level quantification, with an R2 value of 0.98 for GENIE-seq compared to 0.91 for Accel-NGS Methyl-Seq. The correlated methylation values of GENIE-seq across a range of cfDNA input amounts (0.5ng to 100ng) were consistently above 0.96, which was significantly higher than that of Accel-NGS Methyl-Seq (0.80). Importantly, GENIE-seq demonstrated excellent robustness with no potential interferents impacting its performance.

Conclusions

We developed GENIE-seq, which introduces gentle enzymatic conversion of DNA and compact “one-tube” workflow to improve the DNA template usage and reduce potential biases. This sensitive and robust sequencing method holds significant potential for applications in methylation-based liquid biopsies.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Shanghai Weihe Medical Laboratory Co. Ltd.

Funding

Shanghai Weihe Medical Laboratory Co. Ltd.

Disclosure

All authors have declared no conflicts of interest.