- 期刊
以NGS技術分析粒線體DNA甲基化與年齡相關性之研究
The Research of the Correlation Between Mitochondrial DNA Methylation and Age Using NGS Technology
摘要
llumina Miseq定序平台是現今最先進定序平台之一，其特色在於同時定序多股DNA序列，因此，次世代定序（next-generation sequencing, NGS）技術十分適合應用於DNA甲基化分析。目前已有多篇研究指出DNA甲基化可作為預估年齡的參考，實務上年齡係確認身分的重要資訊之一，例如無名屍或嫌犯年齡，皆影響偵查機關的辦案方向。本所每年約有400件無名屍案件，多數死者年齡不詳。因此，本研究藉由NGS技術，分析不同年齡死者法醫大腦檢體之粒線體DNA甲基化情形，藉此評估死者年齡。本研究以NGS技術分析25件法醫檢體，進行bisulfite sequencing，並逐一檢視粒線體鹼基甲基化情形，分析年齡與粒線體DNA甲基化之間的關聯性。本研究篩選粒線體DNA上20個具有顯著甲基化差異位點，計算每一個位點之甲基化程度，再依決定係數分成3大類R^2 ＞ 0.1、0.1 ＞ R^2 ＞ 0.05及R^2 ＜ 0.05，最後篩選出粒線體DNA上4個位點（3886、3485、8954及11381）作為評估年齡參考，其中3886、8954及11381位點之決定係數R^2 ＞ 0.3，則相關係數R約為0.55；另3485位點之決定係數R^2 =0.2358，則相關係數R約為0.48。因此，當R值為0.3 ≦ |R| ＜ 0.7，表示甲基化程度與年齡具有中度相關性。本研究將粒線體3886、3485、8954及11381位點，依目前所得迴歸線方程式，分別計算上述各位點之預測年齡，並與實際年齡做比較，初步發現未知樣本1及2，實際年齡與預測年齡分別差距-13歲及+2歲。未來希望藉由此方法，深入探討更多案件，得到更精確的分析結果，提供檢察機關及法醫師進行年齡估計之參考。
並列摘要
llumina Miseq is one of the most advanced next-generation sequencing (NGS) platforms. It is characterized by the simultaneous sequencing of multiple strands of DNA. Therefore, NGS technology is very suitable for DNA methylation analysis. Many studies have indicated that DNA methylation can predicte the human age. Age is one of the important information for human identification and also effects the criminal investigation. In our institute, there are about 400 unidentified bodies per year and some cases can not identify the age. Consequently, this study collected 25 forensic autopsy cases and analyzed mitochondrial DNA from human brain tissues. The experiments were implemented NGS-based methylation sequencing using whole-genome bisulfite sequencing method and tried to analysis the correlation between mitochondrial DNA methylation and age. Our team selected 20 significant sites of mitochondrial DNA and calculated the methylation degree of each site. The results were clustered into the following three groups based on coefficient of determination: R^2 ＞ 0.1, 0.1 ＞ R^2 ＞ 0.05 and R^2 ＜ 0.05. The 4 sites (3886, 3485, 8954, and 11381) were selected as a reference to estimate the age. There are 3 sites (3886, 8954 and 11381) where the coefficient of determination (R^2) is greater than 0.3, and the correlation coefficient (R) is about 0.55. There is one site (3845) where the coefficient of determination (R^2) is 0.2358, and the correlation coefficient is about 0.48. Therefore, when the R value is 0.3 ≦ |R| ＜ 0.7, it means that the degree of DNA methylation is moderately related to age. In this study, we estimated age of two cases based on the current regression line equations in mitochondrial DNA sites (3886, 3485, 8954 and 11381). The difference between the chronological age and the predictably age of unknown sample 1 and sample 2 is -13 years old and +2 years old, respectively. In the future, we hope that this method can be utilized to provide more information and analysis more cases. This method can provide a reference for the procurator institutes and medical examiners to estimate age of forensic evidence.