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16 Jun 2021
A validation of Illumina EPIC array system with bisulfite-based amplicon sequencing
The Illumina Infinium® Methylation EPIC Bead Chip system (in the future EPIC array) is the current gold standard for detecting DNA methylation at the genome-wide level. The inevitable conclusion of using EPIC arrays for hypothesis development or pilot research is to verify methylation candidates and extend them in a larger cohort in a targeted way. As a result, a precise smaller-scale, focused method is required, which yields data at the individual CpG level akin to the EPIC array. Researchers investigated the potential of bisulfite-based amplicon sequencing (BSAS), an alternate DNA methylation detection approach, to confirm CpG sites found in EPIC array investigations.
The EPIC array system and BSAS were able to identify differential DNA methylation at CpG sites to a great degree. When the amount of change by EPIC array was more than 5%, BSAS correlated less well with EPIC array data, suggesting that the poorer specificity at more significant differential methylation levels is due to the PCR amplification that BSAS needs. However, our findings indicate that BSAS has several benefits over the EPIC array: BSAS amplifies a 500-bp area of the genome surrounding a CpG of interest, allowing analysis of additional CpGs in the region that may not be present on the EPIC array, and assisting in the discovery of new CpG sites and differentially methylated areas of interest. We conclude that BSAS is a useful exploratory tool for certain genomic areas not currently covered by the array system.
DNA Methylation
DNA methylation, for example, is an epigenetic alteration that regulates gene expression and has the potential to cause phenotypic alterations. When a methyl group is covalently added to the C5 position of a DNA molecule's cytosine ring by a methyltransferase enzyme, the resultant changed cytosine is referred to as 5-methylcytosine (5mC). The majority of DNA methylation in mammals occurs at CpG dinucleotides. CpG sites are single modified cytosine residues found mostly in promoter regions of the genome, which are known for their high CpG density.The surrounding environment significantly impacts DNA methylation at CpG sites; variables such as cigarette smoking, alcohol use, diet, stress, and age can all affect. Alterations in DNA methylation are linked to phenotypic alterations, and methylation changes can potentially contribute to disease pathology in rare cases.
The evaluation of differential DNA methylation in humans, particularly epigenome-wide association studies (EWAS), is a blossoming topic due to these relatively new results. Due to their resilience and precision, high-throughput array technologies are a preferred alternative for EWAS. The Illumina Infinium® MethylationEPIC array (hereinafter referred to as the ‘EPIC array') measures methylation at 850,000 different CpG sites, which, while still a small percentage of the total number of CpG sites in the genome (28 million), represents a wide range of sites that provide a precise and reliable measurement of methylation at those sites.
Analysing Relationships Between DNA Methylation Alterations
The next step in analysing relationships between DNA methylation alterations in response to a known environmental exposure at a genome-wide level has been made possible by high-throughput array technology. The EPIC array (along with the previous 27k and 450k arrays) is one such technology that allows these DNA methylation alterations to be characterised.Diverse research has advanced our understanding of how DNA methylation can play a role in response to various environmental exposures using these methodologies. The EPIC array is still the most reliable method for measuring DNA methylation. This is because the method's probe-based nature typically yields similar results across research organisations and arrays. When employing the EPIC array to identify differential methylation, researchers discovered a 23 percent difference among cannabis plus tobacco users compared to controls, a finding that is corroborated by previous studies in the field.
Alternative Method
Finally, we opted to confirm EPIC array data by detecting differential methylation at CpG sites using the alternate approach, BSAS. However, probably, BSAS won't replicate the number of alterations shown in the EPIC array system, which might be due to PCR amplification's lack of specificity and addition error rate. However, instead of assessing individual CpG sites, it may examine differentially methylated areas. Due to some parts of the genome being more prone to methylation changes than others, BSAS was able to discover swaths of associated differential methylation at neighbouring CpG sites in certain parts of the genome. According to the findings, BSAS can detect methylation marks like metastable epialleles, which might be markers for illness later in life. Finally, we show that, while BSAS does not yield the same degree of significance as the EPIC array in some cases, it may be utilised as an exploratory tool for certain genomic areas (Noble et al., 2020).If you are searching for a DNA extraction kit for DNA testing, MBP Inc is the right spot for you!
Bibliography Noble, A., Pearson, J., Boden, J., Horwood, J., Gemmel, N., Kennady, M., & Osbrone, A. (2020, May 28). A validation of Illumina EPIC array system with bisulfite-based amplicon sequencing. BioRxiv. https://doi.org/10.1101/2020.05.25.115428
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