Methylation Detection from Sanger Sequencing
Mutation Surveyor includes a unique tool for the detection of DNA Methylation sites. Compatible with DNA sequences generated by all major sequencing systems, Applied BioSystems Prism®, Beckman Coulter CEQ and MegaBACE, this tool offers a unique method for Methylation Detection from Sanger Sequence reads not present in similar programs such as Variant Reporter™, Sequencher® nor DNAStar®.
Methylation of DNA at the C of CpG sites has been associated with X chromosome inactivation, genomic imprinting, embryonic development, human diseases and differential gene expression. Epigenetic mechanisms involving methylation of DNA have been reported in colon cancer. Differential DNA methylation can be used to identify fetal DNA in maternal blood. Bisulfite treatment of DNA followed by sequencing is one method to analyze methylation of DNA. Improving speed and efficiency of sequence trace analysis will further the understanding of DNA methylation and its relationship to biological processes.
The GenBank sequence of the DNA of interest is used as a “ruler” to report nucleotide changes, including methylations and mutations. The methylation function of Mutation Surveyor utilizes the GenBank sequence text or derivatives of the sequence text and converts it into a synthetic reference sequence. This synthetic reference sequence is then physically compared to the sample sequence traces from bisulfite-treated DNA to find nucleotide differences
Peak-by-peak comparison of the derivative and sample traces can be viewed in the Graphic Analysis Display window. Possible methylation sites will be identified in the mutation electropherogram:
Figure 1: Electropherogram of Methylation Analysis
The top panel is the reference trace synthesized from the derivative GenBank file. The blue lines above the text indicate that the GenBank text was C (blue color). The second panel is the sample trace. The third panel is the mutation detection panel. Methylation analysis in Mutation Surveyor also provides reports for printing and reviewing purposes.
Figure 2: Methylation Graphic Display Report
Methylation Graphic Display Report shows a graphic view of the methylated sites and successful conversion sites detected in the project. You can use this report to review where the methylated sites are located when the samples are aligned to the GenBank file.
Figure 3: Methylation Report
Methylation Report lists the samples at the top and each nucleotide position on the left. M=Methylated, S= Successful conversion, and U=Unsuccessful conversion. You can double click on any of the detected sites and the software will zoom into that position in the main analysis window
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Mutation Surveyor Capabilities:
Detection Sensitivity & Accuracy
Somatic Mutation Detection
Hypervariable Region Variant Detection
Custom Reporting Options
Alignment of Reads
Mitochondria DNA Sequence Analysis