The Development of Age Estimation Model for Unidentified Individuals Based on Mitochondrial DNA Methylation Changes

为了进一步研究年龄相关的线粒体基因组DNA甲基化模式，如果可用的话，从同一肋骨供体收集总共10个血液样本（22-94岁）。对所有血液样品进行DNA提取、定量和亚硫酸氢盐转化。对18个肋骨样本进行分析的9个感兴趣区域（ROIs）进行扩增，然后在Miseq平台（Illumina）上进行测序。还对两个甲基化对照（0%和100%）进行测序，以确保测序质量。在Ubuntu上对原始数据进行修剪，通过CLC-Genomics 11（Qiagen）进行比对和差异DNA甲基化调用。因此，测序质量得到了改善，每个ROI的平均深度为3010个读数。两个ROI（OLS和CytoB）与年龄呈弱相关。Cyto 2的一个CpG位点与年龄呈正相关（R= 0.74），其次是12 S（R=0.587）、G11778 A（R=0.534）、Promoter（-）（R=0.334）和16 S（R=0.331）。ND 5的一个CpG位点与衰老呈负相关（R = 0.587），其次是Cyto 1（R=-0.495）。由于样本量小，采集了42份血液样本（21-94岁），并进行了9个ROI的测序，随后进行了与上述相同的工作流程。出乎意料的是，在大多数样品中观察到中等甲基化水平（40%-60%），导致与年龄增加的相关性可以忽略不计。

To further investigate the age-related DNA methylation pattern of mitochondrial genome, a total number of 10 blood samples (22-94 y.o) was collected from the same rib donor if available. All blood samples were subjected to DNA extraction, quantification and bisulfite conversion. The amplification of 9 regions of interests (ROIs), which have been analyzed with 18 rib samples, was performed then sequenced on Miseq platform (Illumina). Two methylated controls(0% and 100%) were also sequenced to ensure the quality of sequencing. Raw data were trimmed on Ubuntu, the alignment and differential DNA methylation calling were performed by CLC-Genomics Workbench 11 (Qiagen).As a result, the quality of sequencing has improved with a mean depth of 3010 reads at each ROI. Two ROIs (OLS and CytoB) showed weak correlations with aging. One CpG site at Cyto2 showed a positive correlation of 0.74 with aging, followed by 12S(R=0.587), G11778A(R=0.534), Promoter(-)(R=0.334)and 16S(R=0.331). While one CpG site at ND5 showed a negative correlation of 0.587 with aging, followed by Cyto1(R=-0.495). Owing to small sample size, 42 blood samples (21-94 y.o) were collected and proceeded to the sequencing of 9 ROIs followed by the same workflow as mentioned above. Unexpectedly, moderate methylation levels (40%-60%) were observed with most samples, leading to negligible correlations with increasing age.