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Bisulfite Sequencing Services

determine DNA methylation status at base pair resolution

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
Heat map of Targeted Next-Gen Bisulfite Sequencing data
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.

Comparison of data generated by Active Motif Epigenetic Services comparing Sanger and Targeted Next-Gen Bisulfite Sequencing methods
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.

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The papers below cite the use of and/or provide additional information about Next-Gen Bisulfite Sequencing Services provided by Active Motif’s Epigenetic Services: