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Bisulfite sequencing is a method used to reveal the methylation status of select genomic regions at base pair resolution. The method relies on sodium bisulfite treatment of double-stranded genomic DNA, which leads to deamination of unmethylated cytosines to uracils, while methylated cytosines remain unchanged. The DNA is amplified by PCR using primers that anneal to the converted DNA, and the amplicons are then sequenced.
Active Motif Epigenetic Services offers two Bisulfite Sequencing options:
- Sanger Bisulfite Sequencing – This option is the more traditional approach that requires cloning of bisulfite-converted amplicons and sequencing of 8 to 16 clones. It is best for small projects that involve only a few samples or amplicons.
- Targeted Next-Gen Bisulfite Sequencing – This option uses Next-Gen sequencing to generate data from thousands of clones. It is better suited for larger projects because the sequencing can be multiplexed to accommodate many samples and amplicons.
To learn more, please give us a call or send us an Epigenetic Services Information Request. You can also download Active Motif’s Epigenetic Services Profile.
| Name | Cat No. | Price | |
|---|---|---|---|
| Bisulfite Sequencing Service: Targeted Next-Gen | 25035 | Request Quote | |
| Bisulfite Sequencing Service: Sanger | 25042 | Request Quote | |
| Active Motif Epigenetic Services Profile |
| Epigenetic Services Sample Submission Form |
| Sample Preparation for DNA Methylation Services |
Active Motif’s Sanger Bisulfite Sequencing Service is the traditional bisulfite sequencing methodology that requires cloning of the bisulfite-converted amplicons followed by colony selection and sequencing of 8 to 16 individual clones. Amplicons are typically 350 to 450 bp in length.
Active Motif’s Bisulfite Sequencing Service includes all steps from primer design and optimization, bisulfite conversion, PCR, cloning and sequencing to final analysis.
Bisulfite sequencing reveals differential methylation in two cell lines.
Active Motif performs custom Bisulfite sequencing on customer selected genomic locations. The assay includes primer design, bisulfite conversion, cloning, sequencing and data analysis. The data above compares the methylation state (empty circle = unmethylated, purple circle = methylated) of a 400 bp region containing 24 CpGs in two different cell lines. Seven clones from each of the cell lines were sequenced.
Active Motif’s Targeted Next-Gen Bisulfite Sequencing Service offers significant advantages over the traditional Sanger-based method, most notably the ability to multiplex several amplicons from multiple bisulfite-converted DNA samples in a single Next-Gen sequencing reaction. Because 100X to 10,000X more data is generated for each cytosine, the resulting data is more statistically significant than Sanger sequencing. The service is an end-to-end assay and includes bisulfite conversion, primer design and testing, PCR amplification, library preparation, DNA sequencing and data analysis.
Advantages of Targeted Next-Gen Bisulfite Sequencing
- More sequencing data results in greater statistical significance
- Use longer amplicons than Sanger Bisulfite Sequencing
- A more cost effective option for large-scale experiments using multiple cell types and/or amplicons
Figure 1: Heat map of Targeted Next-Gen Bisulfite Sequencing data.
MethylCollector Ultra-Seq was performed on 3 samples of interest to identify differentially methylated regions. One identified region was used for Targeted Next-Gen Bisulfite Sequencing on a broader population of 9 samples. This heat map shows the single base pair resolution of the 72 CpGs in this region across the 9 samples.
Figure 2: Comparison of Sanger and Targeted Next-Gen Bisulfite Sequencing data.
DNA from two different cell lines was bisulfite converted using Active Motif’s MethylDetector™ Bisulfite Modification Kit. Bisulfite sequencing primers were designed to amplify a 312 bp amplicon containing 17 CpG dinucleotides. For Sanger sequencing, the amplicons were cloned and 16 colonies from each sample were sequenced. Methylated CpGs are depicted as red boxes and unmethylated CpGs are depicted as blue boxes. For Targeted Next-Gen Bisulfite Sequencing, amplicons were sequenced directly resulting in over 10,000X coverage. The points in the line graph represent the percent methylation detected at each CpG. Quantitative differences in methylation are detectable between the two samples at CpGs 4 through 16.
