Epigenetic changes, such as DNA methylation and other histone modifications, represent a heritable layer of information that regulates how DNA will be transcribed. These changes are essential for understanding gene regulation and expression in many organisms, as epigenetic dysregulation is often associated with disease. Currently, there are three common NGS-based methods for analyzing epigenetics—methyl-seq, ChIP-seq, and ATAC-seq.
Methyl-seq is a powerful tool that 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 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 for and analyze DNA fragments of interest bound to a particular protein. As the analyses of epigenomes transition from cell culture to tissue and primary cells, the amount of post-ChIP DNA is often in the picogram range.
ATAC-seq, an assay for transposase-accessible chromatin sequencing, maps regions of DNA accessible to 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. Kapa Biosystems has a number of solutions designed to optimize library quality for epigenetic workflows. The KAPA Hyper Prep 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 such 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, on the other hand, can be used for amplification of both ATAC-seq and ChIP-seq libraries to deliver improved sequence coverage and reduced bias.
Recorded Workshop: Optimized library generation for epigenomic analysis of ultra-low input samples
Part I: Low-input high-throughput DNA methylation analysis
Previously recorded on October 9, 2015 at ASHG 2015. Presented by Ben Berwick of Emory University. For Research Use Only. Not for use in diagnostic procedures.
Part II: Low-input ChIP-seq
Previously recorded on October 9th at ASHG 2015. Presented by Dr. Christopher Scharer of Emory University For Research Use Only. Not for use in diagnostic procedures.
Part III: Assay for Transposase Accessible Chromatin sequencing (ATAC-seq)
Previously recorded on October 9th at ASHG 2015. Presented by Dr. Christopher Scharer of Emory University. For Research Use Only. Not for use in diagnostic procedures.
- Construct high-quality libraries from FFPE and challenging samples in less than 3 hours
- Highly tolerant of uracil in template strands, enabling accurate and efficient amplification of bisulfite-treated DNA
- Reduced Amplification Bias of GC- and AT-rich Genomes
- Improved sequencing coverage