Abstract
The Illumina Infinium® MethylationEPIC BeadChip system (hereafter EPIC array) is considered to be the current gold standard detection method for assessing DNA methylation at the genome-wide level. EPIC arrays are often used for hypothesis generation or pilot studies, the natural conclusion to which is to validate methylation candidates and expand these in a larger cohort, in a targeted manner. As such, an accurate smaller-scale, targeted technique, that generates data at the individual CpG level that is equivalent to the EPIC array, is needed. Here, we tested an alternative DNA methylation detection technique, known as bisulfite-based amplicon sequencing (BSAS), to determine its ability to validate CpG sites detected in EPIC array studies. BSAS was able to detect differential DNA methylation at CpG sites to a degree which correlates highly with the EPIC array system at some loci. However, BSAS correlated less well with EPIC array data in some instances, and most notably, when the magnitude of change via EPIC array was greater than 5%. Therefore, our data suggests that BSAS can be used to validate EPIC array data, but each locus must be compared on an individual basis, before being taken forward into large scale screening. Further, BSAS does offer advantages compared to the probe-based EPIC array; BSAS amplifies a region of the genome (∼500 bp) around a CpG of interest, allowing analyses of other CpGs in the region that may not be present on the EPIC array, aiding discovery of novel CpG sites and differentially methylated regions of interest. We conclude that BSAS offers a valid investigative tool for specific regions of the genome that are currently not contained on the array system.
Highlights
Epigenetic modifications, such as DNA methylation, play a vital role in regulating gene expression [1] and have the potential to induce phenotypic changes [2,3,4,5,6]
based amplicon sequencing (BSAS) is often used as a standalone method for assessing differential DNA methylation at specific CpG sites, usually because it is more cost-effective than EPIC arrays, and allows analysis of many samples at once, in multiplex
BSAS estimation of differential DNA methylation correlated with differential methylation determined via EPIC array
Summary
Epigenetic modifications, such as DNA methylation, play a vital role in regulating gene expression [1] and have the potential to induce phenotypic changes [2,3,4,5,6]. Given the limitations of pyrosequencing and MS-PCR, here we examine whether BSAS is an accurate EPIC array validation, replication and/or expansion tool for targeted DNA methylation analyses. We asked whether BSAS, after determination of the most appropriate normalisation method, produced the same average methylation values as EPIC arrays, when comparing case data to control data.
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