Quantitative Investigation of Methylation Heterogeneity by Digital Melting Curve Analysis on a SlipChip for Atrial Fibrillation

Methylation is an essential epigenetic modification involvedinregulating gene expression and maintaining genome stability. Methylationpatterns can be heterogeneous, exhibiting variations in both leveland density. However, current methods of methylation analysis, includingsequencing, methylation-specific PCR, and high-resolution meltingcurve analysis (HRM), face limitations of high cost, time-consumingworkflows, and the difficulty of both accurate heterogeneity analysisand precise quantification. Here, a droplet array SlipChip-based (da-SlipChip-based)digital melting curve analysis (MCA) method was developed for theaccurate quantification of both methylation level (ratio of methylatedmolecules to total molecules) and methylation density (ratio of methylatedCpG sites to total CpG sites). The SlipChip-based digital MCA systemsupplements an in situ thermal cycler with a fluorescence imagingmodule for real-time MCA. The da-SlipChip can generate 10,656 dropletsof 1 nL each, which can be separated into four lanes, enabling thesimultaneous analysis of four samples. This method's clinicalapplication was demonstrated by analyzing samples from ten healthyindividuals and twenty patients with atrial fibrillation (AF), themost common arrhythmia. This method can distinguish healthy individualsfrom those with AF of both the paroxysmal and persistent types. Italso holds potential for broader application in various research andclinical settings requiring methylation analysis.

Automated summary

Methylation is an essential epigenetic modification involved in regulating gene expression and maintaining genome stability. Current methods of methylation analysis face limitations of high cost, time-consuming workflows, and difficulty in accurate heterogeneity analysis and precise quantification. A droplet array SlipChip-based digital melting curve analysis (MCA) method was developed for accurate quantification of both methylation level and density. This method has been demonstrated to distinguish healthy individuals from patients with atrial fibrillation and holds potential for broader application in various research and clinical settings requiring methylation analysis.