整合參考基因體重組技術與序列遞迴修正方法進行次世代基因體序列分析

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研究生：	孫敬倫 Sun, Jing-Lun
論文名稱：	整合參考基因體重組技術與序列遞迴修正方法進行次世代基因體序列分析 IRAP: An Iterative Reference-Guided Assembly Pipeline for Next Generation Sequencing DNA-seq analysis
指導教授：	唐傳義 Tang, Chuan-Yi 王雯靜 Wang, Wen-Ching
口試委員:	唐傳義 Tang, Chuan-Yi 王雯靜 Wang, Wen-Ching 劉明麗 Liou, Ming-Li
學位類別：	碩士 Master
系所名稱：	電機資訊學院 - 資訊工程學系 Computer Science
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	50
中文關鍵詞：	序列重組、基因註解、組序、貼序
相關次數：	點閱：2 下載：0
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這篇論文中，我們的目標是希望能整合貼序(aligner)及組序(assembler)兩種方法來提高最後重組完序列的準確性及提升已被註解的基因數量。目前序列重組主要有兩種方式:貼序及組序，兩者最大的差別在於貼序必須要給定基因體序列(reference)。兩種方法各有其優缺點，貼序的方式可以利用較少的資源來找到高相似度的序列，但是貼序結果的好壞會受限於使用的基因體序列跟定序片段(reads)的相似度。組序的優點是可以不需要給定基因體序列，所以不會受限於要有已知且相似的基因體序列，但是組序的結果會因定序片段的錯誤率及演算法上給定的特定值而有相當的差異。
基因註解上我們會使用已知的基因序列來做註解，並針對在基因體序列上註解的基因來利用定序片段來偵測是否有因抗藥性而產生的突變。在有偵測到突變的基因裡，我們會用蛋白質結構的變化及基因功能(gene function)來做篩選，並利用蛋白質與蛋白質之間的交互作用(protein-protein interactions)來篩選出最有可能的變異基因來做生物驗證。驗證方面我們使用聚合酵素鏈鎖反應(polymerase chain reaction)、基因剔除(gene knockout)等級相關生物實驗來做驗證，利用這些實驗可以驗證我們組的基因體序列的準確性，另一方面也可以去驗證在基因上找到的突變是否跟抗藥性相關。

Background:
For discovering antibiotic resistant genes in functional genomics, comprehensive variant detection between target genome and template genome is important especially variant on an annotated genes. While Next-generation sequencing (NGS) of high throughput DNA sequencing (DNA-seq) has emerged as a powerful technology to conquer these problems, the success of each assembled tool are dependent upon the availability and quality of detected genes.
Results:
Here, we describe IRAP (An Iterative Reference-guided Assembly Pipeline for next generation sequencing DNA-seq analysis), a pipeline that assemble reads into contigs and predict gene models by designing an iterative re-sequencing and integrating de novo assembly of DNA-seq data. We have simulate five distinct Helicobacter pylori lineages generated by ART by IRAP assembly pipeline and applied to real data of three Helicobacter pylori isolate pairs and compared the results to the several existing assembly tools. The contigs produced by IRAP has highly accurate (over 99%) and reconstruct more full-length genes for the majority of the existing reference gene sets (100%). All results showed that IRAP can match or outperform than current popular assemblers.
Conclusions:
These results demonstrate that the IRAP pipeline is able to reconstruct more full-length genes accurately and has more chance to discover potential genes which associate to antibiotic-resistant directly or indirectly.

CHAPTER I：Introduction
1    Background    .......................................................................................... 1
2    Alignment    .......................................................................................... 1
3    Assembly    .......................................................................................... 2
4    IRAP        .......................................................................................... 3
CHAPTER II：Materials and Methods 
1    Simulated Short Reads        .................................................................. 4
2    Next Generation Sequencing of Taiwanese Clinical H. pylori Genomes                         .......................................................................................... 4 
3    Reference Genome Data        .................................................................. 5 
4    Confirmatory PCR-based Sequencing of Short H. pylori DNA Tracts                            .......................................................................................... 5 
5    De Novo Assembly Tools    .................................................................. 6 
6    IRAP: A Custom-built Bioinformatics Pipeline for the Automated Annotation and Analysis of Sequence Data 
6.1    Phase 1:    Re-sequencing and Iterative Reference Sequence Modification        .................................................................. 6
6.2    Phase 2:    Deriving Contigs by Separating Conserved Regions and Their Overlapping Regions Construction                                    .................................................................. 7
6.3    Phase 3:    De Novo Assembly, Redundant Contigs Filtering and Super-contigs Construction         ........................................ 8
        2.6.4    Phase 4:    Base Refinement of Super-contigs        .................. 8
7 Assembly Accuracy Evaluation     ........................................................ 9
CHAPTER III：Results 
1     The IRAP Assembly Pipeline        ........................................................ 11
2     Quality Evaluation of Assembled Super-contigs: Simulated Sequences from Five H. pylori Lineages            ........................................................ 13
3    Quality Evaluation of Assembled Super-contigs: Real World Data of Six Taiwanese H. pylori Clinical Strains        ........................................ 14
4    Evidence Based on Gene Coverage Shows the Close Relationship between Taiwanese and Japanese H. pylori Strains    ........................................ 14
5    Computational Complexity and Comparison to the Other Assemblers
5.1    Comparing with Other Assembly Tools Using Simulated Data    ........................................................................................ 15
5.2    Comparing with Other Assembly Tools Using Real World Data        ........................................................................................ 16
6    Gene Model Annotations    ................................................................ 16
CHAPTER IV：Conclusions 
    Conclusions        ........................................................................................ 18
Reference            ........................................................................................ 19
APPENDIX             ........................................................................................ 22

                                

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