Applications
ChIP-seq/Methylation/Epigenetics
Overview:
Chromosomal DNA is frequently methylated or interacts with various proteins as a means of modifying gene expression. The Genome Sequencer FLX System's high-throughput and long, accurate reads enable a comprehensive and high-resolution view of the epigenome. In methylation experiments, the long reads allow a highly sensitive view of the methylation status of multiple CpG sites in a contiguous read. Read lengths longer than the rotation of DNA around a histone provide a high resolution view of protein/DNA interactions in ChIP experiments.
Publications:
- Mavrich et al. Nucleosome organization in the Drosophila genome. Nature 453: 358-62. May 15, 2008.
- Korshunova et al. Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA. Genome Research 18: 19-29. January 2008.
- Korbel et al. Paired-End Mapping Reveals Extensive Structural Variation in the Human Genome. Science 318: 420-6. October 19, 2007.
Click here to see a full list of ChIP-seq/Methylation/Epigenetics publications.
Application Notes:
Detection and Quantitation of Methylation Patterns using Amplicon Sequencing (PDF)
Data Analysis Tools:
GS Amplicon Variant Analyzer Software
How it Works:
ChIP-Sequencing
With the Genome Sequencer FLX System it is possible to combine the chromatin immunoprecipitation (ChIP) with high-throughput sequencing. For this application, cells are treated with formaldehyde in order to crosslink genomic DNA with the DNA-binding proteins. The transcription factors of interest (or other DNA binding proteins) are immunoprecipitated together with the corresponding DNA using transcription factor-specific monoclonal antibodies. After removal of the proteins, paired-end tags of the remaining DNA fragments can be generated and sequenced in a high-throughput manner.
Methylation patterns
Applying a standard bisulfite-treatment procedure, the Genome Sequencer FLX System enables the quantitative characterization of the methylation state of each CpG dinucleotide in a given target genomic sequence. For this approach DNA is treated with sodium bisulfite, resulting in the deamination of unmethylated cytosines to uracils, while methylated cytosines remain unchanged.
Figure 1: Treatment of genomic DNA with sodium bisulfite leading to the deamination of unmethylated cytosines to uracil.
Subsequent PCR amplification of the converted cytosine (now uracil) results in the substitution of thymine for the uracil. Comparison of the sequence obtained from the bisulfite-treated amplicon to the published sequence using the GS Amplicon Variant Analysis Software enables identification of any differential methylation.