Influences gene expression through chemical modifications that do not alter DNA sequence.
Epigenetic changes, such as DNA methylation and histone modifications, represent a heritable layer of information that regulates DNA transcription. In many organisms, these changes are essential for understanding gene regulation and expression. Epigenetic dysregulation, such as the methylation of DNA (CpG), modifications of histones, binding of microRNAs to block translation, post-transcriptional silencing by short interfering RNA (siRNA), and modification of chromatin structure by noncoding RNAs (ncRNAs), are associated with several diseases, including cancer. Epigenetics studies can unveil variations in expressed phenotypes, X-chromosome inactivation and transcription errors.
Both array-based and next generation sequencing (NGS)-based methods are used for studying epigenetic modifications. There are three common NGS-based methods available for epigenetic analysis: methyl-seq, ChIP-seq, and ATAC-seq.
Methyl-seq investigates the methylation status of the genome with single-nucleotide resolution. This method employs bisulfite treatment, which converts cytosine residues into uracil, while methylated residues are left unmodified. Several methyl-seq strategies have been developed including whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS), which enriches for CpG islands.
ChIP-seq combines chromatin immunoprecipitation (ChIP) with NGS to identify binding sites of DNA-associated proteins throughout the genome, and is routinely used to map histone modifications and transcription factors. This method relies on targeted antibody selection to enrich DNA fragments of interest bound to a particular protein.
ATAC-seq, an assay for transposase-accessible chromatin sequencing, determines regions of chromatin accessibility and maps DNA binding proteins to identify active promoters, enhancers, and other cis-regulatory elements. This method has transformed the analysis of gene regulation by allowing the generation of sequencing libraries with as few as 50,000 cells.
Because epigenetic analyses often involve ultra-low input DNA, the construction of high-quality libraries from limited material is critical. Roche Sequencing Solutions has a number of solutions designed for target enrichment, library preparation and optimized library quality for epigenetic workflows. The KAPA HyperPrep Kit is ideally suited for both ChIP-seq and methyl-seq applications as it enables a higher yield of adapter-ligated library and lower amplification bias. This translates to higher library diversity, lower duplication rates, and more uniform coverage, particularly for low-input samples. For methyl-seq studies, the KAPA HiFi Uracil+ HotStart DNA Polymerase is essential for the amplification of bisulfite-converted libraries due to its tolerance to uracil residues. KAPA HiFi HotStart ReadyMix can be used for amplification of both ATAC-seq and ChIP-seq libraries to deliver improved sequence coverage and reduced bias.