組裝與分析轉錄體短序列定序資料應用於東方果實蠅抗藥性之研究

Abstract

近年來，隨著次世代定序技術(Next Generation Sequencing, NGS)的蓬勃發展與成熟，大量降低定序成本與時間，使得許多物種的基因體以及轉錄體定序也開始蓬勃發展。近年來，逐漸有專家學者們針對短序列組裝提出各種不同的演算法，而這些基因體以及轉錄體的序列分析與註解也是更加急迫的研究議題。 本篇論文中，主要分成兩大部分：第一部分為組裝工具的比較，利用三種組裝工具針對東方果實蠅(Bactrocera dorsalis)的cDNA短序列進行組裝，組裝好的序列首先與NCBI上已公布的20條東方果實蠅完整mRNA序列進行比較，再進一步與相近物種-黃果蠅(Drosophila melanogaster)的蛋白質序列比對，藉由驗證序列品質來討論組裝工具的優缺點。第二部分為組裝結果的分析，利用東方果實蠅的對福木松殺蟲劑的三種不同的狀態，感性、抗性以及復性的組裝結果進行分析，針對其轉錄體之基因功能分析(Gene Ontology)、基因表現量差異(Differential Gene Expression)分析、抗藥相關酵素預測(Enzyme Prediction)以及單核苷酸變異(Single Nucleotide Mutation)等四個部分做分析以及討論。

In recent years, with the dramatically advance of the next generation sequencing (NGS), the sequencing cost and the time required for experiments and analysis have been largely saved. Thus, completely sequencing new organisms become popular and universal. Recently, many assembly algorithms have been developed. In addition, the demands of automated gene annotation and the analysis of genome and transcriptome data are urgently desired. This study is divided into two parts. The first part is concerning the comparison of different assembly tools. In this research, the Bactrocera dorsalis short reads were assembled sing three well-known assembly tools. After that, we aligned the assembled sequences with 20 Bactrocera dorsalis complete mRNA sequences from NCBI and the protein sequences of Drosophila melanogaster, the closet model species that can be found currently. The advantages and disadvantages of assembly tools were discussed by evaluating the quality of assembled sequences. The second part is the analysis of assembled sequences. In this part, we assembled the short reads from three different conditions with respect insecticide: sensitive, resistant, and recovered, respectively. In the end, this study characterizes the assembled sequences in terms of Gene Ontology (GO), differentially expressed genes (DEGs), enzyme prediction, and single nucleotide mutation.