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NGS-BSP

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The Introduction of NGS-BSP

DNA methylation strongly affects chromatin structure and the regulation of gene expression. For many years, Bisulfite Sequencing PCR (BSP) has served as the “gold standard” for measuring DNA methylation of specific regions. BSP has high reliability and accuracy, and can accurately determine the methylation status of each CpG site in the fragment. The Next Generation Sequencing-based Bisulfite Sequencing PCR (NGS-BSP) can find more precise methylation modification information, and is applicable to some genes that are known methylated detection regions, and is also suitable for exploring significant methylation regions of genes.

Treatment of DNA with bisulfite converts cytosine residues to uracil but leaves 5-methylcytosine residues unaffected. The BSP primers were designed for PCR amplification of target regions. After purification of the PCR product, a PCR library was constructed, followed by next-generation sequencing. Finally, the sequencing reads can be used to map the original sequence, the methylation site and number were counted, and the level of methylation was analyzed.

Key Features and Advantages

  • Quickly detect all methylation sites
  • High throughput, High accuracy, Simple and intuitive results
  • More cost-effective and time-saving than original BSP if testing a lot of samples

Project Workflow

In brief, BSP primers were designed using the online MethPrimer software. Genomic DNA (1 ug) was converted using the ZYMO EZ DNA Methylation-Gold Kit (ZYMO) and one twentieth of the elution products is used as templates for PCR amplification. For each sample, BSP products of multiple genes were generated, pooled equally and subjected to adaptor ligation. Barcoded libraries from all samples were sequenced on the Illumina Hiseq platform using paired-end 150 bp strategy.

NGS-BSP Workflow

NGS-BSP Workflow

Sample Requirements

  • gDNA amount: ≥ 1 μg;
  • DNA concentration ≥ 30 ng/μl
Sequencing Strategy

  • Illumina HiSeq, PE150.
  • Depth of coverage ≥ 100x.

CD Genomics offers consultation with you at every stage of the process to ensure that you make the most use of your data. We offer professional assistance from planning experiments to sequencing and further data analysis. If you have any requirements do let us know and we will be there to assist you. We’d love the opportunity to work with you. Please contact us for more information and a detailed quote.

References
Pan X, Gong D, Nguyen DN, et al. Early microbial colonization affects DNA methylation of genes related to intestinal immunity and metabolism in preterm pigs[J]. DNA Research, 2018.

Zhang S, Shen L, Xia Y, et al. DNA methylation landscape of fat deposits and fatty acid composition in obese and lean pigs[J]. Scientific Reports, 2016, 6:35063.

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