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

为了进一步研究线粒体基因组与年龄相关的DNA甲基化模式，总共收集了10份血液样本(22-94岁)，如果有的话，来自同一肋骨捐赠者。对所有血样进行DNA提取、定量和亚硫酸氢盐转化。对18个肋骨标本进行9个感兴趣区(ROI)的扩增，并在MiSeq平台(Illumina)上进行测序。还对两个甲基化对照(0%和100%)进行了测序，以确保测序质量。在Ubuntu上对原始数据进行修剪，用CLC-Genology Workbench 11(Qiagen)进行比对和差异DNA甲基化调用，测序质量得到改善，每个ROI的平均阅读深度为3010个阅读。两个感兴趣区(OLS和CytoB)与年龄呈弱相关。Cyto2的一个CpG位点与年龄呈正相关，依次为12S(R=0.587)、G11778A(R=0.534)、启动子(-)(R=0.334)和16S(R=0.331)。而ND5的一个CpG位点与年龄呈负相关，相关系数为0.587，其次是细胞因子1(R=-0.495)。由于样本量小，采集了42份血液样本(21-94岁)，并按照上述相同的工作流程对9个ROI进行了测序。出乎意料的是，在大多数样本中观察到了中等甲基化水平(40%-60%)，导致与年龄增长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.