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系統識別號 U0026-2007202021504500
論文名稱(中文) 提升免疫基因組探索之效率
論文名稱(英文) Improving the efficiency of immune repertoire exploration
校院名稱 成功大學
系所名稱(中) 生物科技與產業科學系
系所名稱(英) Department of Biotechnology and Bioindustry Sciences
學年度 108
學期 2
出版年 109
研究生(中文) 洪聖柔
研究生(英文) Sheng-Jou Hung
學號 Z28011027
學位類別 博士
語文別 英文
論文頁數 81頁
口試委員 指導教授-劉宗霖
召集委員-曾大千
口試委員-鄭順林
口試委員-張文綺
口試委員-林以行
口試委員-施純傑
口試委員-陳倩瑜
口試委員-張偉嶠
中文關鍵字 免疫基因組  T 細胞受體(TCR)  V(D)J 重組  5’ cDNA末端快速擴增(5’ RACE) 
英文關鍵字 Immune repertoire  T-cell receptor (TCR)  V(D)J recombination  5’ rapid amplification of cDNA ends (5’ RACE) 
學科別分類
中文摘要 免疫系統中的T細胞透過其受體識別特異性外來抗原,並激活免疫反應。T細胞受體 (TCR) 基因在V(D)J重組過程中,選擇並連接各種V、(D) 和J基因,所形成多樣的TCR序列能夠反映免疫系統的功能。為抓取各種TCR基因來研究免疫基因組,常用的多重PCR方法透過多種引子來放大所有可能的重組基因,但它有引子偏差的問題。要避免引子偏差可以使用5’ cDNA末端快速擴增 (5’ RACE) 的方法。然而,5’ RACE會產生非規則重組的TCR序列。現有的序列比對工具會誤把非規則重組的序列當成規則序列而導致錯誤的註解。因此我們開發了TRIg來正確分析5’ RACE數據。 TRIg將TCR序列與整個免疫基因比對,而不僅僅與V(D)J區域比對。有了準確的序列比對工具,我們在一個健康人樣本中比較多重PCR和5’ RACE方法,發現5’ RACE方法的可重複性比較高,而且偵測到的免疫基因組較均勻,顯示其較無引子偏差的優勢。另外,我們改良了5’ RACE方法,降低非規則重組序列的比例,使得大部份資料能有效用於免疫基因組的分析。這些成果提供有效率的5’ RACE實驗和計算分析流程以研究免疫基因組。
英文摘要 In the adaptive immune system, T cells are able to recognize a variety of foreign antigens because their T cell receptors (TCRs) appear in diverse structures, which are the results of complex V(D)J recombination of TCR genes. The recombined TCR gene sequences of all T cells can thus be used to characterize immune repertoire. To capture all possible V(D)J recombinations, a popular approach is applying multiple primers that target all possible V and/or J regions of TCR genes for amplification. This multiplex PCR (mPCR) approach, however, usually introduces primer bias. To avoid primer bias, a 5’ rapid amplification of cDNA ends (5’ RACE) approach can be used to ampify TCR genes. In the 5’ RACE data, however, both regularly and non-regularly recombined TCR sequences exist, and the later of which could not be used to characterize immune repertoire. Current tools may mistake non-regular TCR sequences as regular and report false V(D)J annotations. In this thesis, we developed a new computational tool, TRIg, to correctly handle both regular and non-regular TCR sequences in the 5’ RACE data. To promote the 5’ RACE approach, we further studied the difference between mPCR and 5’ RACE approach. We found that 5’ RACE achieved a higher consistency and captured more VJ recombination events than mPCR, suggesting less primer bias of the 5’ RACE approach. Finally, we improved a 5’ RACE method to reduce the proportion of non-regular TCR sequences via carefully controlling size of the PCR amplicons. This increased the fraction of useful data from <40% to >80%. With all these efforts, we provide an efficient experimental and computational pipeline for studying immune repertoire.
論文目次 Chinese Abstract (中文摘要) I
Abstract II
Acknowledgements V
Table of Contents VI
Contents of Tables IX
Contents of Figures X
Contents of Appendices XII
Abbreviation List XIII
Chapter 1 Research Background 1
1-1 Immune system and T cell receptor 1
1-2 V (D) J recombination 1
1-3 Application of immune repertoire 3
1-4 Current methods and problems 4
1-5 Research objectives 7
Chapter 2 Materials and Methods 9
2-1 Materials and methods for aim I 9
2-2 Materials and methods for aim II 11
2-3 Materials and methods for aim III 13
Chapter 3 Results 17
3-1 Developing a computational tool for analyzing 5’ RACE data 17
3-2 Comparing multiplex PCR and 5’ RACE approach 25
3-3 Improving the efficiency of a 5’ RACE experimental protocol 29
Chapter 4 Discussion 31
4-1 Primer bias in Multiplex PCR 31
4-2 Reproducibility of our 5’ RACE protocol 31
4-3 The role of non-regular sequence 33
4-4 Experimental conditions 34
4-5 Conclusion 35
References 36
Tables 41
Figures 51
Appendices 73
Related Paper Publications 81
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