So Moon Hyun, Lee Ji Eun, Lee Hwan Young
Department of Forensic Medicine, Seoul National University College of Medicine, Seoul, South Korea.
Institute of Forensic and Anthropological Science, Seoul National University College of Medicine, Seoul, South Korea.
Electrophoresis. 2024 May;45(9-10):906-915. doi: 10.1002/elps.202300185. Epub 2024 Mar 15.
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.
使用单碱基延伸（SBE）的靶向亚硫酸氢盐测序可用于通过法医实验室基因分析仪上的毛细管电泳来测量DNA甲基化。已经报道了几种使用该方法的准确年龄预测模型。然而，使用具有不同软件设置的不同基因分析仪会产生不同的甲基化值，从而导致年龄预测出现重大误差。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）为每种基因分析仪类型构建新的年龄预测模型；（4）构建可应用于所有类型基因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，先前报道的调整方程被用于血液/唾液DNA年龄预测标记（ELOVL2、FHL2、KLF14、MIR29B2CHG/C1orf132和TRIM59）。通过在三种类型的基因分析仪（3130、3500和SeqStudio）的结果之间绘制多项式回归线，为精液DNA年龄预测标记（TTC7B、LOC401324/cg12837463和LOC729960/NOX4）生成了新的方程。使用相同的方法获得了使用11个对照DNA样本的调整方程。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。对于与基因分析仪无关的模型，使用对照DNA来制定校准每种基因分析仪数据偏差的方程，并使用校准后的体液DNA数据进行线性回归分析。在比较结果中，特定于基因分析仪的模型显示出最高的准确性。然而，通过偏差调整的与基因分析仪无关的模型也提供了准确的年龄预测结果，表明在存在多种限制的情况下可将其用作替代方法。
