臺灣博碩士論文加值系統

研究背景與目的
隨著台灣人口老年化程度越來越高加上檢查技術越來越精進，每年罹癌人數一直在節節攀升，現在每五分鐘就有一名台灣人罹癌，癌症時鐘已比十年前快了1.5倍。隨著標靶藥物的興起，對癌細胞的基因資訊在臨床上已被大量需求。次世代定序技術是一個運用在基因檢測上非常有力的工具，隨著該技術的高速發展和普及化，其所花費的成本也越來越低，因此次世代定序技術目前已非常合適用於臨床檢驗上面。本篇論文主要探討以Ion AmpliSeq Cancer Hotspot Panel v2是否適合做為臨床之使用，測試其在臨床檢體上所呈現的效能。

研究方法
從大腸癌病患取得之癌組織塊檢體，立即抽取核酸。選取先前已經過高解析解離分析過15個癌症相關基因的檢體。此檢體在相同濃度的狀況下進行三人六次之重複試驗，使用的是Ion AmpliSeq Cancer Hotspot Panel v2試劑組及Ion Proton平台，並依原廠建議流程進行操作。最後由次世代定序技術得到的資訊再以桑格定序法進行驗證，並與之前高解析解離分析資料互相比對，由這些資
料進行其效能驗算與證實。

研究結果
六次試驗平均資料輸出結果為2890萬鹼基，可用讀值數為25萬。其中一次與整體偏離較大，其可用讀值數僅15萬，導致其無訊號位點數也較多。每次試驗所得到的基因突變位點可偵測總量皆大於原本所試劑組宣稱的2855個位點，在無訊號的分析中，平均有1.2%的位點是不可被採用的，其中無訊號比例占最高的基因為RB1(25%)、CDH1(14%)。在資料分析中，全部出現有突變的位點共20個，其中13個每次都能偵測的到，但是在這13個位點中，僅有八個被桑格定序法驗證為真，其餘五個雖然每次都有被偵測到且其覆蓋深度及所占比率皆不低的情況下，仍是一個偽陽性的結果。在桑格定序法驗證及比對先前高解析解離分析
資料，專一性為99.86%，再現性為94.59%。

結論
本研究測試的檢驗套組，其再現率及專一性皆能有不錯的表現，但是在偽陽性率的部分高達了38.5%，對其在檢驗上的效能大大打了折扣。在本實驗中觀察到了即使有高再現率、覆蓋深度及突變比率的突變位點，仍有可能是一個偽陽性的結果，因此我們建議，臨床在使用次世代基因定序技術時，所有的關鍵性/致病性突變位點一定得在使用第二種方法進行驗證，避免造成誤判的情形產生。

Introduction
Next-Generation Sequencing (NGS) is a powerful tool to analyze genome changes. With falling cost, NGS is more suitable to be a diagnosis tool in clinical applications. However, the performance of diagnostic tests must be verified and validated in a clinical diagnostic laboratory. We evaluated the applicability of the Ion AmpliSeq Cancer Hotspot Panel v2 using Ion Proton Sequencer for screening 50 cancer-related genes in fresh tumor tissues.

Methods
DNA was extracted from the fresh tissue biopsy of colorectal tumor. Use the Ion AmpliSeq Cancer Hotspot Panel v2 and Ion Proton Sequencer for screening hotspot regions of 50 oncogenes and tumor suppressor genes. The same sample was detected repeatedly by this panel for six times. We compared the concordances and differences data between six tests, and all mutation variants were confirmed by Sanger
sequencing. We evaluated the clinical validation of this panel.

Results
The average Ion Proton sequencing output per run was 28.9 megabases with 0.25 million sequencing reads. The reproducibility of this panel counted from six tests was 94.42%. In total, the Ion Proton detected 20 expected mutation variants, and these variants were related to 14 genes. In these 20 variants, 13 variants were detected in every test, 2 variant were detected in three times and twice respectively, and 5 variants only appeared once. All of the variants appeared less than four times were confirmed to be false, but 5 of 13 variants which were detected in every test were also confirmed to be false by Sanger sequencing. Two false variants* were caused by the defect of method due to homopolymers (>6 identical bases). The true mutation variants were all from novel mutations (not include in designed hotspots), but three mutation variants from hotspots were all false. In addition, the high coverage variant# was also proved to be false. From these findings, it’s hard to distinguish the variant is true or false only by few quality data. We only calculated the mutation variants detected in six times, but the false variant rate was still 38.5%.

Conclusions
This panel can simultaneously screen 50 cancer-related genes covering 2,855 COSMIC mutations with a low input of DNA .It''s a convenient and powerful tool with reduced time and cost of genetic analysis to implement in clinical diagnosis. We revealed the risk of false variants with high reproducibility and high coverage. Therefore, we suggest that it''s necessary to verify any variant for the clinical use of the Ion Ampliseq cancer hotspot panel v2.

目錄

英文摘要 1
中文摘要 2
誌謝詞 3
目錄 4
第一章 前言 6
第一節 研究背景 6
第二節 研究目的 7
第二章 研究方法 8
第一節 研究材料 8
2.1.1 檢體種類及來源 8
2.1.2 核酸萃取 8
2.1.3 建庫 8
2.1.4 檢體條碼化 9
2.1.5 乳液聚合酶鏈鎖反應 9
2.1.6 Ion Proton定序儀 9
2.1.7 桑格定序法 10
第二節 研究設計 10
2.2.1 檢體選取 10
2.2.2 重覆試驗 10
2.2.3 數據統整及驗證 10
第三節 統計方法 10
2.3.1資料分析 10
第三章 研究結果 11
第一節 描述性統計分析 11
3.1.1 定序品質總論 11
3.1.2 突變子的統計分析 11
3.1.3 全新突變的出現率 11

3.1.4 無訊號分析 13
3.1.5 資料的正確性 16
3.1.6 資料的再現性與準確性 16
第二節 推論性統計分析 22
3.2.1 各次操作間的差距 22
3.2.2 無訊號的探討 22
3.2.3 特異度與靈敏度 22
3.2.4 再現性與準確性探討 23
第四章 討論 24
第一節 結果討論 24
第二節 其他相關性討論 24
第三節 研究限制 25
第五章 結論與建議 26
第一節 結論 26
第二節 建議 26
參考文獻及附錄 27

圖表目錄

表一 定序品質總表 11
表二 全部突變熱點在各基因的數量 12
表三 新突變點在各基因的數量 13
表四 無訊號(NOCALLS)的數量與機率統計 14
表五 突變子驗證資訊 15

圖一 桑定序法驗證資料 18
圖二 各突變點的覆蓋深度 21

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