The impact of methodology on the reproducibility and rigor of DNA methylation data

Detlev Boison,Joyce Ohm,Jonathan D Geiger,Theresa Lusardi,Farah D Lubin,Kai Guo,Richard Sanchez,Junguk Hur,Susan A Masino,David N Ruskin

doi:10.1038/s41598-021-04346-w

Abstract

Epigenetic modifications are crucial for normal development and implicated in disease pathogenesis. While epigenetics continues to be a burgeoning research area in neuroscience, unaddressed issues related to data reproducibility across laboratories remain. Separating meaningful experimental changes from background variability is a challenge in epigenomic studies. Here we show that seemingly minor experimental variations, even under normal baseline conditions, can have a significant impact on epigenome outcome measures and data interpretation. We examined genome-wide DNA methylation and gene expression profiles of hippocampal tissues from wild-type rats housed in three independent laboratories using nearly identical conditions. Reduced-representation bisulfite sequencing and RNA-seq respectively identified 3852 differentially methylated and 1075 differentially expressed genes between laboratories, even in the absence of experimental intervention. Difficult-to-match factors such as animal vendors and a subset of husbandry and tissue extraction procedures produced quantifiable variations between wild-type animals across the three laboratories. Our study demonstrates that seemingly minor experimental variations, even under normal baseline conditions, can have a significant impact on epigenome outcome measures and data interpretation. This is particularly meaningful for neurological studies in animal models, in which baseline parameters between experimental groups are difficult to control. To enhance scientific rigor, we conclude that strict adherence to protocols is necessary for the execution and interpretation of epigenetic studies and that protocol-sensitive epigenetic changes, amongst naive animals, may confound experimental results.

Highlights

Detailed epigenome analyses applied to translational disease models typically find dozens to thousands of potential epigenetic modifications at gene regions, and the process of identifying causal factors is unavoidably challenging and time-consuming
A recent study examined genomewide DNA methylation levels without matching experimental protocols in three different animal models of epileptogenesis, each performed in a different laboratory, and found no meaningful common changes in DNA methylation associated across the three models, which led the authors to conclude that there was no mechanistic overlap among models[10]
SAS-Sprague Dawley male rats were purchased from vendors (Charles River and Envigo) nearest to our three project sites; Legacy Research Institute in Portland, Oregon (Site #1), Trinity College in Hartford, Connecticut (Site #2), and the University of Alabama at Birmingham in Birmingham (UAB), Alabama (Site #3)

Summary

Introduction

Detailed epigenome analyses applied to translational disease models typically find dozens to thousands of potential epigenetic modifications at gene regions, and the process of identifying causal factors is unavoidably challenging and time-consuming. Adding further to this complexity are observations of major differences in epigenetic signatures among different models of the same disease. To begin to address whether baseline experimental analysis of DNA methylation can be influenced by interlaboratory protocol-related confounds, we sought to compare DNA methylation marks in control wild-type tissue collected from three different laboratories. Hippocampal tissues were harvested and examined for DNA methylation and associated gene expression differences across the three laboratories (Fig. 1), minimizing protocol differences, and matching variables such as vendor, age, rat strain, and tissue processing method for analysis

Methods

Results

Conclusion