Batch effects and pathway analysis: two potential perils in cancer studies involving DNA methylation array analysis.

Area of Science:

• Epigenetics and Cancer Research
• Genomics and Bioinformatics

Background:

• DNA methylation microarrays are widely used for epigenetics research in cancer.
• Current analysis methods for these arrays are still evolving and not universally adopted.

Purpose of the Study:

• To investigate potential issues in DNA methylation microarray analysis: batch effects and inappropriate use of pathway analysis software.
• To evaluate the impact of chip-specific batch effects and the application of gene expression analysis tools on methylation data.

Main Methods:

• DNA samples were analyzed twice on the Illumina Infinium 450 K HumanMethylation Array to assess batch effects.
• Simulations were performed using random CpG sites from the 450 K array and Ingenuity's IPA software for pathway analysis.

Main Results:

• A significant portion of differentially methylated sites identified were attributed to batch effects, with few consistent findings between runs.
• Pathway analysis software generated numerous spurious associations between randomly selected methylation data and diseases/biological functions.

Conclusions:

• Chip-specific batch effects can mimic genuine differential methylation findings.
• Pathway analysis software designed for gene expression arrays can produce misleading results when applied to DNA methylation data, emphasizing the need for careful data handling and appropriate analytical tools.