Epigenetic Studies
ChIP Sequencing
By combining chromatin immunoprecipitation (ChIP) assays with sequencing, ChIP sequencing (ChIP-Seq) is a powerful method for identifying genome-wide DNA binding sites for transcription factors and other proteins. Following ChIP protocols, DNA-bound protein is immunoprecipitated using a specific antibody. The bound DNA is then coprecipitated, purified, and sequenced.
The application of next-generation sequencing (NGS) to ChIP has revealed insights into gene regulation events that play a role in various diseases and biological pathways, such as development and cancer progression. ChIP-Seq enables thorough examination of the interactions between proteins and nucleic acids on a genome-wide scale. We use Illumina based sequencing technology methods for this.
MeDIP Sequencing
MeDIP-sequencing involves fragmenting the DNA into various lengths and then using a 5mC antibody to target the methylated fragments. Although this is a relatively low-cost method that can sequence the whole-genome DNA, it has poor resolution, which makes it difficult to precisely identify the methylated site. Advantages of methylation sequencing:
- Discover methylation patterns of CpG, CHH, and CHG regions across the human genome
- View methylation at practically every cytosine in the genome across most species with whole-genome bisulfite sequencing (WGBS), a genome-wide approach
- Capture full sample diversity with small amounts of DNA
- Cover emerging regions of interest in the human genome identified by ENCODE, FANTOM5, and the Epigenomics RoadMap Consortium with targeted methylation sequencing
Whole Genome Bisulphite Sequencing
The NGS-driven bisulfite sequencing is useful for providing a global view of epigenomes and also for identifying genomic loci with different levels of DNA methylation. Next-generation sequencing technology has enabled genome-wide analysis of 5mC nucleotides at single-nucleotide resolution. Previous methods for whole-genome bisulfite sequencing (WGBS) yielded reduced genomic representation due to required DNA shearing, ligation of methylated sequencing adapters, and bisulfite conversion of unmethylated cytidine residues prior to sequencing. This heritable epigenetic mark remains stable during cell division and acts as a form of cellular memory, regulating various cellular activities such as transcription and chromosomal stability, and plays a crucial role in embryonic development, genomic imprinting and X-chromosome inactivation. In particular, aberrant DNA methylation has been found to be associated with various complex human diseases such as cancer, autoimmune diseases, metabolic disorders, type-2 diabetes and obesity.
Reduced Representation Bisulphite Sequencing
Reduced representation bisulfite sequencing (RRBS) is an efficient and high-throughput technique for analyzing the genome-wide methylation profiles on a single nucleotide level. It combines restriction enzymes and bisulfite sequencing to enrich for areas of the genome with a high CpG content. Due to the high cost and depth of sequencing to analyze methylation status in the entire genome. DNA methylation has been associated with gene silencing, tissue differentiation, genomic imprinting, X chromosome inactivation, phenotypic variation, and possibly disease susceptibility [9–13]. Aberrant DNA methylation is implicated in several diseases and has a well-established role in tumorigenesis.
