Targeted bisulfite sequencing using single‐base extension (SBE) can be used to measure DNA methylation via capillary electrophoresis on genetic analyzers in forensic labs. Several accurate age prediction models have been reported using this method. However, using different genetic analyzers with different software settings can generate different methylation values, leading to significant errors in age prediction. To address this issue, the study proposes and compares four methods as follows: (1) adjusting methylation values using numerous actual body fluid DNA samples, (2) adjusting methylation values using control DNAs with varying methylation ratios, (3) constructing new age prediction models for each genetic analyzer type, and (4) constructing new age prediction models that could be applied to all types of genetic analyzers. To test the methods for adjusting values using actual body fluid DNA samples, previously reported adjusting equations were used for blood/saliva DNA age prediction markers (ELOVL2, FHL2, KLF14, MIR29B2CHG/C1orf132, and TRIM59). New equations were generated for semen DNA age prediction markers (TTC7B, LOC401324/cg12837463, and LOC729960/NOX4) by drawing polynomial regression lines between the results of the three types of genetic analyzers (3130, 3500, and SeqStudio). The same method was applied to obtain adjustment equations using 11 control DNA samples. To develop new age prediction models for each genetic analyzer type, linear regression analysis was conducted using DNA methylation data from 150 blood, 150 saliva, and 62 semen samples. For the genetic analyzer‐independent models, control DNAs were used to formulate equations for calibrating the bias of the data from each genetic analyzer, and linear regression analysis was performed using calibrated body fluid DNA data. In the comparison results, the genetic analyzer‐specific models showed the highest accuracy. However, genetic analyzer‐independent models through bias adjustment also provided accurate age prediction results, suggesting its use as an alternative in situations with multiple constraints.

中文翻译：

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处理遗传分析仪特定 DNA 甲基化测量的策略

使用单碱基延伸 (SBE) 的靶向亚硫酸氢盐测序可用于在法医实验室的遗传分析仪上通过毛细管电泳测量 DNA 甲基化。已经报道了使用这种方法的几种准确的年龄预测模型。然而，使用具有不同软件设置的不同遗传分析仪可能会产生不同的甲基化值，从而导致年龄预测出现重大错误。为了解决这个问题，该研究提出并比较了以下四种方法：（1）使用大量实际体液DNA样本调整甲基化值，（2）使用具有不同甲基化比率的对照DNA调整甲基化值，（3）构建新的年龄预测每种遗传分析仪类型的模型，以及（4）构建可应用于所有类型遗传分析仪的新年龄预测模型。为了测试使用实际体液 DNA 样本调整值的方法，之前报道的调整方程用于血液/唾液 DNA 年龄预测标记（埃洛夫2,FHL2,KLF14,MIR29B2CHG/C1orf132， 和修剪59）。为精液 DNA 年龄预测标记生成了新方程（TTC7B,LOC401324/cg12837463， 和LOC729960/NOX4）通过在三种类型的遗传分析仪（3130、3500 和 SeqStudio）的结果之间绘制多项式回归线。使用 11 个对照 DNA 样品应用相同的方法获得调整方程。为了为每种基因分析仪类型开发新的年龄预测模型，使用来自 150 份血液、150 份唾液和 62 份精液样本的 DNA 甲基化数据进行了线性回归分析。对于与遗传分析仪无关的模型，使用对照 DNA 制定方程来校准每个遗传分析仪数据的偏差，并使用校准的体液 DNA 数据进行线性回归分析。在比较结果中，遗传分析仪特定模型显示出最高的准确性。然而，通过偏差调整，独立于遗传分析仪的模型也提供了准确的年龄预测结果，表明它可以作为具有多重约束的情况下的替代方案。