Evaluation of the pooled sample method in Infinium Methylation EPIC Bead Chip array by comparison with individual samples (original) (raw)

2023, Research Square (Research Square)

Background: The pooled sample method is used in epigenomic research and expression analysis and is a cost-effective screening approach. Evaluation of the pooled sample method in epigenomic studies is performed using the Illumina In nium Methylation 450 K BeadChip array; however, subsequent reports on the updated 850 K array are lacking. A previous study demonstrated that the methylation levels obtained from individual samples were accurately replicated using pooled samples but did not address epigenome-wide association study (EWAS) statistics. The DNA quanti cation method, which is important for the homogeneous mixing of DNA in the pooled sample method, has since become uorescencebased, and additional factors need to be considered including the resolution of batch effects of microarray chips and the heterogeneity of the cellular proportions from which the DNA samples are derived. In this study, four pooled samples were created from 44 individual samples, and EWAS statistics for differentially methylated positions (DMPs) and regions (DMRs) were conducted for individual samples and compared with the statistics obtained from the pooled samples. Results: The methylation levels could be reproduced fairly well in the pooled samples. This was the case for the entire dataset and when limited to the top 100 CpG sites, consistent with a previous study using the 450 K BeadChip array. However, considering a dataset of 1,000 randomly extracted CpG sites, the statistical results of the EWAS for the DMP by individual samples were not replicated in pooled samples. Qualitative analyses highlighting methylation within an arbitrary candidate gene were replicable. Focusing on chr 20, the statistical results of EWAS for DMR from individual samples showed replicability in the pooled samples as long as they were limited to regions with a su cient effect size. Conclusions: The pooled sample method replicated the methylation values well and can be used for EWAS in DMR. This method is cost-effective and can be utilized for screening by carefully understanding the features for its effective points and disadvantages of the pooled sample method and combining it with candidate gene analyses. Background Research on epigenomes and their interaction with genetics which re ect the "nurture and environment" aspect has shifted the focus from solely studying genetic sequences in various elds (1). Epigenomic research, initially dominated by cancer research (2), has recently expanded to include studies on the relationship between various phenotypes, such as aging (3-7), nutrition and diet (8), environmental pollutants (9, 10), immunity (11, 12), neurological disorders (13), psychiatric disorders (14-17), and psychological traits (18, 19), and is expected to increase further (20). Converse to data-driven genome-wide association analysis, hypothesis-driven candidate gene analysis is the primary approach for conducting epigenomic studies. However, studies that focus on the results of candidate gene analysis and candidate gene-by-environment interactions alone for understanding complex traits without replication experiments have been becoming outdated (21). As genome-wide data,