Introduction
DNA hydroxymethylation is an important epigenetic modification that regulates gene expression and plays an important role in neural differentiation and cancer. However, based on the traditional bisulfite method, 5-hmc and 5-mc cannot be distinguished. Spark gene technology has established the chemical oxidation method combined with bisulfite conversion sequencing technology (oxbs SEQ), which can not only accurately detect DNA methylation and exclude the influence of DNA hydroxymethylation, but also accurately detect DNA hydroxymethylation with single base resolution at the same time.
BS transformation cannot distinguish between 5mC and 5hmc
In traditional bisulfite sequencing, 5hmc becomes CMS after bisulfite treatment. CMS is still read as C base in sequencing, so it cannot distinguish between 5mC and 5hmc.
Principle of oxbs SEQ Technology
Oxbs SEQ oxidizes 5hmc to 5FC, which can be converted to u by bisulfite, thus achieving accurate detection of 5mC; At the same time, the accurate detection of 5hmc can be achieved by comparing with the results of conventional bisulfite.
Technical advantages
1. A new "gold standard" for DNA methylation detection
2. genome wide single base detection of DNA hydroxymethylation modification
3. multiple standards verify high oxidation efficiency and high bisulfite conversion rate
4. low experimental preference and high repeatability (R ² >0.98)
5.可满足多种测序应用需求：简化基因组氧化甲基化测序（oxRRBS），目标区域氧化甲基化测序（Target-oxBS）
Experimental strategy
Information analysis
Technical parameter
| Sample requirements | Project cycle |
| Sample type: DNA sample without degradation and pollution; Sample requirements: ≥ different samples have different requirements, human samples ≥ 3 μ g; Sample concentration: ≥ 30 ng/ μ l sample purity: od260/280=1.8~2.0 | The standard operation cycle of less than 15 samples is about 45 working days. The large sample quantity projects need to build the warehouse in batches, and the project cycle needs to be determined with the technical support personnel. |
Case analysis
Case：
Oxbs single base detection of 5mC and 5hmc levels in murine embryonic stem cells
Booth, M. J., et al. (2012). Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336(6083): 934-937.
Background：
With the discovery of 5hmc in mammalian genome, the traditional bisulfite sequencing can not accurately distinguish the modification difference between 5mC and 5hmc. In the sequencing results of traditional BS, the modification level of 5mC is actually the combination of 5mC and 5hmc signals. It is urgent to establish an experimental technology to accurately distinguish the two.
Methods：
Oxbs sequencing technology was used to detect and quantify DNA methylation and hydroxymethylation in mouse embryonic stem cells.
Conclusion:
In this study, the experimental technology of chemical oxidation combined with bisulfite treatment was established for the first time. This technology first oxidizes 5hmc to 5FC, which can then be converted into u by bisulfite, thus eliminating the signal interference of 5hmc to 5mC, achieving the purpose of accurately detecting 5mC in the genome. Using this technology to study mouse embryonic stem cells, it was found that 5hmc has a high content in CGI related transcriptional regulatory regions and Line1 elements, indicating that it may play an important role in epigenetic reprogramming.
Percentages of 5mC and 5hmc in different elements of mouse embryonic stem cell genome
