Genetic/epigenetic Assays
Massively parallel sequencing technology lays the foundation for the construction of epigenomics. Creative Biolabs provides key sequencing-based methods used in the analysis of epigenomes.
- Bisulfite Sequencing
5-methyl-cytosine (5mC) and its oxidized derivatives are measured on a genome-wide basis using enrichment and transformation methods followed by massively parallel sequencing. Bisulfite conversion provides a quantitative measurement of 5mC but does not distinguish 5hmC. Antibody enrichment provides qualitative measurements of 5mC and 5hmC. The bisulfite converted or enriched DNA is purified, library constructed and clonally sequenced. A special algorithm is needed to align the bisulfite conversion readings with the reference genome.
- Chromatin Immunoprecipitation Sequencing
The genomic location of the modified histones is measured genome-wide by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). Histones can be released from the genome by ultrasound, enzymatically digested, or inserted through a transposon. If sonication is used, chromatin must first be chemically cross-linked. After histone release, specific chemical modifications are enriched by immunoabsorption. Purification of enriched histone-associated DNA, which can be purified for high-throughput sequencing, provides complete information on specific DNA-protein interactions and binding sites.
- 3D Chromatin Capture
The genomic location of long-range chromatin exposure is measured genome-wide by large-scale parallel sequencing of DNA fragments generated by adjacent ligation. Complete chromatin cross-linking physically connects adjacent genomic distal nucleosomes in 3-dimensional space. The cross-linked chromatin is enzymatically digested, and the resulting DNA ends are labeled with biotin and ligated adjacently. The ligated DNA is sheared by sonication or enzymatic digestion and the linked ligation is enriched by streptavidin pull-down. The resulting DNA is purified, library constructed and clonally sequenced. Chromosome conformation capture (3C) techniques provide the location of DNA fragments that interact based on their proximity to three-dimensional (3D) space. To measure whole-genome chromatin interactions, a single experiment typically requires 500 million sequences read.
- Determination of Open Chromatin
Large-scale parallel sequencing of DNA fragments released from intact chromatin by transposon insertion, enzymatic digestion or sonication can measure the location of open chromatin in the genome-wide range. The obtained DNA fragment is subjected to size selection or phenol-chloroform extraction to eliminate nucleosome-associated DNA. The resulting DNA is purified, library constructed and sequenced, and aligned to the reference genome. Sequencing requirements depend on experimental parameters and resolution requirements, and range from 10 s to 100 s millions of fragments per sample.
