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研究生: 李宜謙
Lee, Yi-Chien
論文名稱: 線蟲比較基因體學
Comparative genomics of free-living nematodes
指導教授: 蔡怡陞
Tsai, Isheng
口試委員: 蔡怡陞
Tsai, Isheng
莊樹諄
Chuang, Trees-Juen
楊姍樺
Yang, Shan-Hua
顧銓
Ku, Chuan
駱乙君
Luo, Yi-Jyun
口試日期: 2023/09/19
學位類別: 博士
Doctor
系所名稱: 生物多樣性國際研究生博士學位學程
Taiwan International Graduate Program on Biodiversity
論文出版年: 2023
畢業學年度: 112
語文別: 英文
論文頁數: 97
英文關鍵詞: Free-living nematode, nematode community, nematode genomics, whole genome amplification, de novo genome assembly
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202301786
論文種類: 學術論文
相關次數: 點閱:59下載:0
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  • Over 70% of nematode species are free-living. They are found in a wide variety of habitats and are essential for maintaining microenvironments. However, studies on nematodes have mainly focused on parasites and terrestrial free-living. Free-living nematodes in aquatic ecosystems represent 50% of all nematode species but are poorly understood. Recently, studies of marine free-living nematodes have focused on their potential to address nematode phylogeny questions and explore the evolution of nematode parasitism and adaptation to extreme environments. My Ph.D. aims to sequence the genome of a marine free-living nematode to reconstruct nematode phylogeny and compare it with published nematode genomes to investigate nematode genome diversity. During the sampling process, I analyzed the nematode community around the northern coast of Taiwan. It is important to note that marine nematode species are not amenable to axenic culture under laboratory conditions. To alleviate this issue, I utilized multiple displacement amplification (MDA) and Smartseq2 to amplify nanograms of genomic DNA and mRNA from one individual, respectively. We evaluated the bias of this protocol using Caenorhabditis elegans. While reduced genome coverage was detected in repetitive regions, we generated assemblies that covered 98% of the reference genome using long-read sequences produced with Oxford Nanopore Technologies (ONT). Annotation of the sequenced transcriptome and available assemblies showed that gene predictions were more precise, comprehensive, and contained fewer false positives than de novo transcriptome assembly methods. Our results demonstrate the robustness of MDA in combination with ONT, paving the way for the study of genome diversity in the phylum Nematoda and beyond.

    Chapter One: Introduction 1 1.1 Nematodes 1 1.2 Insufficient taxon sampling in nematodes 3 1.3 Comparative genomics in nematodes 5 1.4 Nematodes mitochondria genome 6 1.5 Whole genome amplification 7 1.6. Objectives 9 Chapter Two: Nematode diversity in the North Taiwan Shores 10 2.1 Introduction 10 2.2 Methods and Materials 11 2.2.1 Sample collection 11 2.2.2. Morphology identification 12 2.2.3 Metabarcoding 12 2.2.4 Data process and analysis 12 2.3 Results 12 2.3.1 Nematode morphological and metabarcoding analysis 12 2.3.2 Nematode diversity in north Taiwan shore 15 2.3.3 Nematode and bacteria community 18 2.4 Discussion 21 2.4.1 Morphology and metabarcoding method on nematode community 21 2.4.2 Nematode distribution in north Taiwan seashore 21 Chapter Three: Whole Genome Amplification Enables Single Worm Sequencing 24 3.1 Introduction 24 3.2 Methods and Materials 26 3.2.1 Single worm DNA extraction 26 3.2.2 Whole genome amplification 26 3.2.3 Genomic DNA library preparation, sequencing and assembly 27 3.2.4 Single worm RNA transcriptome sequencing and assembly 31 3.2.5 Genome annotation 32 3.2.6 Decontamination 33 3.3 Results 33 3.3.1 Whole genome amplification facilitates sufficient DNA for long-read sequencing from single nematodes 33 3.3.2 Whole genome amplification disparity in repetitive regions 33 3.3.3 Presence of palindromic sequences after whole genome amplification 40 3.3.4 Longer T7 endonuclease digestion time increase ONT sequencing performance 42 3.3.5 Complete genome assemblies from amplified sequences 44 3.3.6 High quality annotations from a single nematode genome and transcriptome 46 3.4 Discussion 49 Chapter Four: De Novo Assembly Of Free-Living Nematodes 55 4.1 Introduction 55 4.2 Methods and Materials 56 4.2.1 Single worm DNA extraction 56 4.2.2 Whole genome amplification 57 4.2.3 Genomic DNA library preparation, sequencing and assembly 57 4.2.4 Single worm RNA transcriptome sequencing and assembly 63 4.2.5 Genome annotation 64 4.2.6 Decontamination 64 4.2.7 Phylogenomics of nematodes 65 4.3 Results 68 4.3.1 Whole genome amplification facilitates sufficient DNA for long-read sequencing from single nematodes 68 4.3.2 Genome characteristics of free-living nematodes 68 4.3.3 Mitochondral genome of free-living nematodes 78 4.3.4 Enoplia is sister to the rest of the nematode classes 81 4.4 Discussion 84 Chapter Five: Conclusion 86 Reference 88

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