故障排除和最佳实践指南

Methylation arrays are common platforms for analyzing 5-methylcytosine. The Infinium™ MethylationEPIC BeadChip and Infinium™ HumanMethylation450 BeadChip (commonly referred to as the EPIC and 450K arrays respectively) as well as the recently launched Infinium™ Mouse Methylation BeadChip from Illumina^®all utilize Zymo Research’s bisulfite conversion technologies to distinguish 5mC from unmodified cytosines. In addition to the test probe sets, the arrays include bisulfite conversion quality control probes. In some instances, the analysis software will flag samples for low bisulfite conversion efficiency. Possible causes for these warnings are low bisulfite conversion efficiency, low DNA input/quality, and chip failure.

验证的协议

Infinium™甲基化阵列的唯一批准和验证的Bisulfite转换套件是EZ 德赢vwin体育平台入口DNA甲基化试剂盒（目录号码：D5001那D5002那D5004). It is critical to follow the Illumina^®推荐的孵育方案（16个循环为95℃，30秒，50℃，60分钟）。可以在相应的BiSulfite转换协议附录中找到其他详细信息。虽然其他亚硫酸氢盐转化套件将导致高质量的亚硫酸氢盐转化DNA，但仅由illumina支持使用验证套件处理的样品^®。

Along with using the validated bisulfite conversion kits, it is important to follow the rest of the BeadChip protocol as written, including the downstream software analysis. Illumina^®定期审查分析软件和探测性能的性能。使用最新的软件和清单文件以确保下游分析正确。

DNA投入和质量

The amount and quality of genomic DNA used in these assays is critical to success. For best results, DNA should be as intact and high quality as possible. The arrays rely on a signal to noise ratio to determine the conversion efficiency. When low input or low quality DNA is used, the signal to noise ratio is much lower and can affect the reliability of the bisulfite conversion control signals.

手动协议的所需最小DNA为250 ng，自动协议为1000 ng。应使用DSDNA特定方法如Picogreen定量基因组DNA^®or Qubit^®。nanodrop.^®or other spectrophotometric methods are not recommended for quantification due to issues with successfully distinguishing DNA from RNA. RNase treatment can also help ensure that DNA quantification is accurate.

当使用与来自FFPE样品中分离的DNA或其他降解的DNA样品一起使用时，强烈建议使用500ng DNA输入或更高。另外，由于使用较小的洗脱量的能力，建议使用单柱二硫酸氢盐转换（与96孔板）进行降解样品。然后应使用illumina Infinium™FFPE DNA修复试剂盒（如手册中描述的阵列）进行双硫酸盐转换的FFPE DNA。应使用整个样品。

低亚硫酸氢盐转换效率

Bisulfite conversion can be impacted by a variety of factors. The quality of the CT Conversion Reagent is critical for successful conversion. It is recommended to prepare the reagent fresh before each conversion if possible. Otherwise, the prepared reagent should be stored according to the guidelines listed in the protocol. The conversion reagent should not be exposed to light or oxygen any more than necessary. If processing 96-well plates, a multichannel pipette should be used and the prepared conversion reagent should be added last to the plate.

转换应在具有加热盖的热循环仪中进行。样品和转化试剂应彻底混合（未观察到的混合线）并在将管放入热循环仪之前完全旋转。如果管道未完全旋转或未加热盖子，则沉淀可以在PCR管的盖子周围形成。孵育后，应在转移样品时避免观察到的任何沉淀，因为它可以含有未转化的DNA。

The desulphonation incubation should be stopped at 15 minutes (20 minutes is the absolute maximum). Leaving the desulphonation buffer on the column longer than the recommended time can result in additional degradation to the sample.

强烈建议使用Bisulfite转换质量控制检查。可以使用各种方法进行评估亚硫酸氢盐转化。双硫酸氢盐转化的DNA可以通过QPCR量化或使用Nanodrop上的RNA设置量化^®（或类似的分光光度法）。亚硫酸氢盐转化后的预期DNA产率约为70-80％。该量化步骤将有助于确保在下游加工中恢复足够的亚硫酸氢盐转化的DNA。可以通过菌落Sanger测序或通过探针/塔克曼评估亚硫酸氢盐转换效率^®测定。在罕见的情况下，样品纯度可能负责低亚硫酸氢盐转化效率。在这些情况下，建议重新提取或进一步清理。

芯片失败

On occasion, chip failures may occur. If multiple samples on a single chip have low bisulfite conversion efficiency as determined by the BeadArray Controls Reporter, this might indicate a chip failure rather than a bisulfite conversion issue. In these instances, re-running leftover bisulfite-converted sample on a new chip can resolve the issue. To further troubleshoot, a post-bisulfite conversion/ pre-array QC is helpful to determine whether to rerun the samples.

结论

Methylation arrays such as the 450K and EPIC array are helpful tools for analyzing methylation changes in a variety of human and mouse samples. Whenever a sample is flagged for low bisulfite conversion, it is best to confirm that the following is true for your project:

• Validated and updated protocols were used
• DNA尽可能高，完整。使用较高的DNA输入量用于碎片/低质量样品
• Bisulfite conversion was performed according to best practices

Following these simple steps will help determine the reason for the bisulfite conversion probe failure.

2月11日2021年