DNA Methylation Analysis via Bisulfite Sequencing
DNA methylation has been shown to be involved in a diverse array of cellular functions and diseases, including gene expression, differentiation, genomic imprinting, X chromosome inactivation, transposon silencing, regulation of chromatin structure and genome integrity, carcinogenesis, aging, and stem cell differentiation. DNA methylation patterns are dynamically regulated during development by a suite of epigenetic regulators, and the influence of the environment. In addition, DNA methylation changes are generally accepted as one of the most common molecular changes in human tumors.
The gold standard in analyzing DNA methylation is by chemical treatment of DNA using bisulfite. Bisulfite treatment converts cytosine to uracil while 5-methy cytosine is resistant to the conversion. After the treatment, genomic DNA is subject to PCR amplification, subcloning, and sequencing. The sequencing result is then compared to the original sequences, and any methylated/unmethylated cytosine is unambiguously determined. As a result, methylated Cs remains as Cs while unmethylated Cs become Ts (see figures below).
We offer comprehensive bisulfite sequencing service, including genomic DNA digestion, bisulfite conversion, target primer design, PCR amplification, subcloning, and random sequencing. For statistical representation of each cytosine in any given sample type, 15-20 individual clones need to be sequenced. Depending on your experimental needs, we can help you design best strategy to address your research questions.
DNA Methylation Profiling via Whole Genome Bisulfite-Seq
Recent advance in DNA methylation technologies has enable profiling of the entire methylome at single base resolution. We provide Bisulfite-seq ready Solexa PE library construction, and whole genome bisulfite sequencing on Solexa GAII platform. We also provide informatics support. Please contact us for more information.
Bisulfite-seq PE library is made by ligating methylated PE (mPE) adaptor to fragmented genomic DNA. The mPE adaptor-ligated genomic DNA is then bisulfite treated twice, resulting in conversion rate of > 99% of unmethylated cytosine. Bisulfite converted library is then recovered by limited cycles of PCR amplification. The final library size of around 200-300 bp is gel purified and quantified via fluorometer. We QC the library quality by random subcloning and sequencing of ~20-30 colonies to check for proper library construction and bisulfite conversion. The QC-ed library is then ready for cluster formation and high throughput Illumina sequencing.
We provide custom data analysis including:
1. Alignment to the reference genome
2. Determine methylation ration for each sequenced cytosine
3. Statistical analysis of differential methylation between two samples or tissue types.
4. Visual distribution of the differentially methylated regions.
5. Other custom analyses.
For more details and for price quote please send inquiry along with your target sequences to firstname.lastname@example.org (email preferred).