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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林宜玲(Yi-Ling Lin) | |
| dc.contributor.author | Sheng-Huai Zheng | en |
| dc.contributor.author | 鄭盛懷 | zh_TW |
| dc.date.accessioned | 2023-03-19T22:54:09Z | - |
| dc.date.copyright | 2022-10-03 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-08-01 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85269 | - |
| dc.description.abstract | 嚴重特殊傳染性肺炎 (COVID-19)是一種由嚴重急性呼吸道症候群冠狀病毒2型 (SARS-CoV-2)引起的傳染性疾病，現今病毒快速的變異及傳播對公共衛生造成嚴重的威脅。透過高通量定序技術 (high-throughput sequencing)可以獲取各種病毒株基因序列及基因表現量資訊，並用於探討基因序列及轉錄組圖譜 (transcriptome profiles)的變異和差異性。在本論文中，為了快速掌握SARS-CoV-2病毒主序列 (consensus sequence) 及其他變異株的基因組資訊，我們建立第三代定序的奈米孔定序技術 (Nanopore Sequencing)，其中包含兩種定序方式，分別為藉由多組引子 (primer)擴增基因全長的增幅式定序 (PCR-tiled sequence)，以及可以維持基因轉錄本 (transcripts)原始分佈的直接RNA定序 (Direct RNA sequence)。首先我們藉比較增幅式定序與其他定序平臺產生的主序列來驗證方法的準確性。在不同SARS-CoV-2病毒株中，與PacBio或Illumina平臺生成的序列相比，奈米孔定序識別出幾乎完全相同的基因特徵。除此之外，奈米孔定序的數據更進一步揭示了主序列層次以下的基因遺傳變異，例如一些比例較少的核苷酸變異在連續繼代培養後，能被保留下來並且逐漸累積成為主序列。藉由直接RNA定序，我們對病毒核酸轉錄本進行全面性的觀測，其中依賴前導轉錄調節序列生成之典型次基因組核酸 (TRS-L dependent canonical subgenomic RNA)佔總基因轉錄組的多數，其次則為非依賴前導轉錄調節序列生成之遠距次基因組核酸 (Distant TRS-L independent subgenomic RNA)。我們發現在遠距次基因組核酸的合成過程中，有顯著比例的轉錄產物是透過病毒基因3'端和核酸位點6800-7000發生大範圍的跳躍和剪接所形成。透過病毒感染的細胞以及基因轉殖小鼠和倉鼠等不同的感染模型，我們證實此類核酸轉錄本的普遍存在。此類轉錄產物在不同宿主中的普遍出現，以及其重組位點在不同病毒株的序列保守性，顯示可能為病毒複製的必要成份。綜上所述，本研究使用奈米孔定序技術觀察到SARS-CoV-2病毒株於繼代培養的序列差異性，也為病毒的次基因組提供新的見解。 | zh_TW |
| dc.description.abstract | Rapid evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) warrants tools to closely characterize the viral genomes at different scales. The advance in sequencing technique enables generating thousands of nucleotide fragments in hours, presenting a promising opportunity to study the genomic architecture and variation of emerging viruses. In this study, we established both the amplicon-based and direct-RNA nanopore sequencing approaches to investigate the genomic composition of SARS-CoV-2. We first validated the amplicon-based approach by comparing the consensus sequences generated by the nanopore pipeline with other sequencing platforms. In different strains, nearly identical genetic signatures were identified by the nanopore method, when compared with the sequences generated by the PacBio or Illumina platform. Data from nanopore sequencing further uncovered minor genetic variants, some of which were subsequently fixed during the serial passage. Next, using the direct-RNA approach we comprehensively characterized the viral RNA transcripts, in which TRS-L dependent canonical subgenomic RNA accounted for most of the RNA species, followed by distant TRS-L independent subgenomic RNA. We identified a notable proportion of viral transcripts (ORF1ab-N) produced through a template switch from 3' end of the genome to a ORF1ab region locating at nucleotide positions 6800-6900 during RNA synthesis. We confirmed the presence of this type of RNA transcript in cultured cells and different animal models including transgenic mice and hamster. The ubiquitousness of these RNA transcripts in different host and the conserved sequences found in the recombination sites suggest biological importance. Collectively, this study provides insights to the SARS-CoV-2 RNA synthesis using nanopore sequencing, and gives hints to disease diagnosis. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T22:54:09Z (GMT). No. of bitstreams: 1 U0001-2707202214253900.pdf: 6544216 bytes, checksum: 2e93d7c4e5b4b652b94119b65912ae28 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 致謝 i 摘要 ii Abstract iii 目錄 iv 表目錄 vii 圖目錄 viii 第一章 緒論 1 1.1 新型冠狀病毒 1 1.1.1 新型冠狀病毒簡介及臨床症狀 1 1.1.2 新型冠狀病毒之複製與轉錄過程 1 1.1.3 新型冠狀病毒次基因組RNA之轉錄機制 2 1.2 奈米孔定序 (Nanopore sequence) 4 1.2.1 第三代定序 (Third-generation sequencing)之簡介 4 1.2.2 增幅式奈米孔定序 (PCR tailing nanopore sequence) 5 1.2.3 直接RNA定序 (Direct RNA sequence) 6 1.3 研究動機 7 第二章 材料與方法 8 2.1 材料 8 2.1.1 病毒株 8 2.1.2 細胞株 8 2.1.3 動物樣本 8 2.1.4 抗體 (Antibodies) 8 2.1.5 試劑與藥品 8 2.1.6 商品化套組 (Commercial kit) 9 2.2 實驗方法 9 2.2.1 增幅式奈米孔定序 9 2.2.2 直接RNA奈米孔定序 10 2.2.3 增幅式目標序列定序 11 2.2.4 西方墨點法 11 2.3 資料量化與分析 12 2.3.1 SARS-CoV-2轉錄本分類 12 2.3.2 序列資料分析 12 第三章 結果 13 3.1 建立增幅式奈米孔定序技術 (Amplicon-based nanopore sequence) 13 3.1.1 建立增幅式奈米孔定序及序列品質管控 (Quality control) 13 3.1.2 藉由不同定序平台的序列比較驗證增幅式奈米孔定序的準確性 13 3.1.3 增幅式奈米孔定序於監測SARS-CoV-2病毒序列之應用 14 3.2 建立直接核酸奈米孔定序 (DRS)技術 16 3.2.1 驗證直接核酸奈米孔定序之準確性 16 3.2.2 探討不同SARS-CoV-2病毒株轉錄組差異 17 3.3 SARS-CoV-2轉錄組譜分析 19 3.3.1 典型次基因組核酸表現量與蛋白質表現量並無直接相關 19 3.3.2 ORF1ab-N型次基因組核酸在不同病毒株中都有存在 19 3.3.3 ORF1ab-N型次基因組核酸於不同動物模式中廣泛存在 20 討論 22 參考文獻 25 附表 30 附圖 35 | |
| dc.language.iso | zh-TW | |
| dc.subject | 嚴重特殊傳染性肺炎 | zh_TW |
| dc.subject | 奈米孔定序 | zh_TW |
| dc.subject | 直接RNA定序 | zh_TW |
| dc.subject | 新型冠狀病毒 | zh_TW |
| dc.subject | Direct RNA sequence | en |
| dc.subject | COVID-19 | en |
| dc.subject | SARS-CoV-2 | en |
| dc.subject | Nanopore sequencing | en |
| dc.title | 藉由奈米孔定序技術研究及分析新型冠狀病毒RNA圖譜 | zh_TW |
| dc.title | Investigation of SARS-CoV-2 RNA profiles by Nanopore Sequencing | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳美如(Mei-Ru Chen),楊宏志(Hung-Chih Yang) | |
| dc.subject.keyword | 奈米孔定序,直接RNA定序,新型冠狀病毒,嚴重特殊傳染性肺炎, | zh_TW |
| dc.subject.keyword | Nanopore sequencing,Direct RNA sequence,COVID-19,SARS-CoV-2, | en |
| dc.relation.page | 49 | |
| dc.identifier.doi | 10.6342/NTU202201776 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-08-01 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2027-07-29 | - |
| 顯示於系所單位： | 微生物學科所 | |
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