Reproducibility of the Infinium methylationEPIC BeadChip assay using low DNA amounts.

Area of Science:

• Epigenetics
• Genomics
• Molecular Biology

Background:

• The Infinium MethylationEPIC BeadChip (EPIC) is a widely used platform for genome-wide DNA methylation profiling.
• Clinical and forensic applications often involve limited DNA quantities, necessitating an understanding of assay performance at low input levels.

Purpose of the Study:

• To evaluate the reproducibility of the MethylationEPIC BeadChip across a range of DNA input amounts.
• To determine the minimum DNA quantity required for reliable DNA methylation measurements using the EPIC array.

Main Methods:

• Serial two-fold dilutions of DNA were prepared and analyzed on the MethylationEPIC BeadChip on two separate days.
• Reproducibility was assessed by comparing β-value distributions and calculating Pearson's correlation coefficients and standard deviations between days.
• The performance was evaluated across DNA amounts ranging from 500 ng down to 16 ng.

Main Results:

• DNA methylation β-value distributions remained largely unaffected by decreasing DNA amounts.
• Median squared Pearson's correlation coefficient (R) for between-day comparisons decreased from 0.994 (500 ng) to 0.957 (16 ng).
• High reproducibility (percentage of probes with standard deviation ≤ 0.1 > 99.4%) was observed for DNA amounts between 125-500 ng, with reduced reproducibility below 63 ng.

Conclusions:

• The MethylationEPIC BeadChip demonstrates high reproducibility for DNA methylation analysis with input amounts of 125-500 ng.
• DNA input amounts of 63 ng or lower result in decreased reproducibility, potentially impacting downstream analysis in clinical and forensic contexts.
• It is recommended to use at least 125 ng of DNA for reliable and reproducible results with the MethylationEPIC BeadChip assay.