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系統識別號 U0026-2607202016240400
論文名稱(中文) 口腔癌變研究之微生物體資料庫暨分析平台
論文名稱(英文) DeManPo: A Database-Embedded Microbiome Analysis Platform for Oral Carcinogenesis Research
校院名稱 成功大學
系所名稱(中) 電機工程學系
系所名稱(英) Department of Electrical Engineering
學年度 108
學期 2
出版年 109
研究生(中文) 陳廷瑜
研究生(英文) Ting-Yu Chen
學號 N26071704
學位類別 碩士
語文別 中文
論文頁數 90頁
口試委員 指導教授-吳謂勝
口試委員-吳哲宏
口試委員-吳梨華
口試委員-陳玉玲
口試委員-劉宗霖
中文關鍵字 16S rRNA  口腔癌  微生物體  標識基因分析  合併分析 
英文關鍵字 microbiome  16S rRNA  joint analysis  oral cancer 
學科別分類
中文摘要 據衛生福利部2016死因統計,口腔癌位居男性國人癌症死亡原因及發生人數第4位,為了想找到致癌的原因及治療方法,微生物體就成了一個良好的分析對象,透過對口腔中的微生物進行定序,再經過多種分析流程來找出在健康人與口腔癌病人的菌群差異。
許多生物學家在經過實驗得到定序資料後,可能會因為如何使用分析套件進行下游分析而感到苦惱,所以一些能提供微生物體分析的平台則應時而出,包括Nephele、Qiita及Microbiome analyst皆提供良好的分析流程,能讓使用者快速且容易的得到下游分析結果。
目前關於研究口腔癌相關的文獻中,大多數文獻所收集的樣本並不充足,這可能會導致無法完整解析口腔中的微生物體,故透過合併採用相同實驗方式的樣本資料來探討口腔微生物體與癌症的關係慢慢被人們所提及,Qiita及Microbiome analyst雖也提供了合併分析的選項,但並未針對口腔癌收集其樣本資料,故本研究收集了至今已發表有關利用16S rRNA來研究口腔癌微生物體的文獻,並從中得到數篇文獻的樣本資料,再透過相同的前處理流程將樣本整理後彙整成資料庫。
經過探討多個分析平台的優缺點,最終建立一分析平台DeManPo (Database-Embedded Microbiome Analysis Platform for Oral Carcinogenesis Research),在獨立分析中能提供使用者優於Qiita及Nephele的分析模組且能比其他平台可加彈性的利用多重Column將樣本分群,合併分析方面,不僅維持獨立分析擁有的分析模組及多重Column分群,亦提供了專門針對口腔癌文獻收集而來的樣本資料庫讓使用者能與同類型的樣本進行合併分析,這是其他平台都未能做到的。
在案例分析中,本研究透過案例一來驗證平台分析流程的正確性,案例二則為實例演練,透過將相同類型的樣本合併分析得到獨立分析時無法發現的結果,雖然透過多種面向的分析來驗證其生物意義,但還是需經過實驗才能證明,不過增加樣本數的目的已然完成。
且相較於Qiita、Nephele能從定序完的資料開始分析以及Microbiome analyst 強大的分析模組,本平台還有很大的進步空間,須持續更新及精進。
DeManPo分析平台網址:
http://cosbi6.ee.ncku.edu.tw/z70180/qiime_filter_default/
英文摘要 According to the statistics of death from the Ministry of Health and Welfare in 2016, oral cancer ranks fourth in the cause of cancer deaths and the number of occur¬rences among Taiwanese male. In order to find the cause and treatment of cancer, mi¬crobiome have become a good analysis object. After sequencing microbiome, and then through a variety of analysis processes to find out the difference between the flora of healthy people and patients with oral cancer. Although there are many plat¬forms for microbial analysis, there is no microbial analysis platform belongs to oral cancer. Therefore, this study provides an analysis platform for oral cancer microbi¬ome, providing users with a database of oral cancer, so that they can perform joint analysis to achieve the purpose of increasing the sample of the same type. And in or¬der to con¬firm that the joint analysis has its contribution, this study conducted case one to ver¬ify. Then case two is an example exercise. By combining and analyzing the same type of samples to obtain results that cannot be found in independent analysis. Although the biological significance is verified through a variety of face-to-face anal¬ysis, it still needs to be proved through experiments, but the purpose of increasing the number of samples already completed.
論文目次 摘 要 I
EXTENDED ABSTRACT III
致謝 VII
目錄 VIII
圖目錄 XII
表目錄 XVI
第一章 緒論 1
1.1 口腔癌 1
1.2 微生物體 2
1.3 微生物體分析 2
1.3.1 標識基因分析 3
1.3.2 全總基因體分析 3
1.3.3 全總轉錄體分析 4
1.4 標識基因分析流程 4
1.4.1 樣本收集 5
1.4.2 16S rRNA擴增子定序 5
1.4.3 產生ASVs / OTUs 7
1.4.3.1 拆分(Demultiplexing) 7
1.4.3.2 去噪(Denoising)與聚類(Clustering) 7
1.4.3.3 聚類(Clustering) 8
1.4.3.4 去噪(Denoising) 8
1.4.4 分析流程 9
1.4.5 16S rRNA相關分析平台 10
1.4.5.1 Microbiome Analyst 10
1.4.5.2 Qiita 11
1.4.5.3 Nephele 11
1.5 研究動機及目的 12
1.5.1 獨立分析 12
1.5.2 合併分析 13
1.5.3 研究目的 13
第二章 建構資料庫 16
2.1 資料收集 16
2.2 資料前處理 18
2.2.1 檔案轉換: SRA  FASTQ 18
2.2.2 檔案轉換: FASTQ  QZA 19
2.2.3 去噪 20
2.3 彙整資料 25
第三章 網頁分析平台 28
3.1 輸入頁 28
3.1.1 資料上傳 29
3.1.2 品質調控 30
3.1.3 樣本挑選 31
3.1.4 樣本分群 33
3.1.5 註解資料庫及聯絡信箱 34
3.2 分析Pipeline 35
3.3 結果頁 36
3.3.1 多樣性分析(Diversity analysis) 37
3.3.1.1 稀疏曲線(Rarefaction curve) 37
3.3.1.2 Alpha多樣性(Alpha diversity) 38
3.3.1.3 Beta多樣性(Beta diversity) 39
3.3.2 物種分析(Taxonomy analysis) 40
3.3.2.1 物種組成分析(Taxonomy profiling) 40
3.3.2.2 核心性分析(Core analysis) 41
3.3.3 物種差異性分析(Differential analysis of taxon) 42
3.3.3.1 T-test 43
3.3.3.2 Wilcoxon rank-sum test 43
3.3.3.3 KS-test 43
3.3.3.4 Kruskal-Wallis test 44
3.3.3.5 ANCOM 44
3.3.3.6 LEfSe 44
3.3.3.7 隨機森林(Random forest) 45
3.3.4 關聯度分析(Correlation analysis) 45
3.3.4.1 SCNIC 46
3.3.5 功能差異性分析(Differential analysis of functionality) 47
3.3.5.1 PICRUSt2 48
第四章 實例探討 49
4.1 實驗一 49
4.1.1 PRJNA352375 50
4.1.1.1 物種組成分析 50
4.1.1.2 多樣性分析 51
4.1.1.3 物種差異性分析 53
4.1.1.4 功能差異性分析 54
4.1.2 PRJEB4953 55
4.1.3 PRJEB18476 55
4.1.4 SRP107079 57
4.1.5 結論 57
4.2 實驗二 58
4.2.1 物種差異性分析 58
4.2.1.1 Genus層級 59
4.2.1.2 Species層級 67
4.2.2 功能差異性分析 76
4.2.3 多樣性分析 79
4.2.3.1 Alpha多樣性 79
4.2.3.2 Beta多樣性 81
4.3 實驗結果 82
第五章 結論與未來展望 84
5.1 結論 84
5.2 未來展望 84
參考文獻 86
附錄 i

參考文獻 [1] J. Bagan, G. Sarrion, and Y. Jimenez. Oral cancer: clinical features, Oral oncology, vol. 46, no. 6, pp. 414-417, 2010.
[2] Rob Knight, Alison Vrbanac, Bryn C. Taylor, Alexander Aksenov, Chris Callewaert, Justine Debelius, Antonio Gonzalez, Tomasz Kosciolek, Laura-Isobel McCall, Daniel McDonald, Alexey V. Melnik, James T. Morton, Jose Navas, Robert A. Quinn, Jon G. Sanders, Austin D. Swafford, Luke R. Thompson, Anupriya Tripathi, Zhenjiang Z. Xu, Jesse R. Zaneveld, Qiyun Zhu, J. Gregory Caporaso, and Pieter C. Dorrestein. Best practices for analysing microbiomes, Nature Reviews Microbiology, vol. 16, no. 7, pp. 410-422, 2018.
[3] John M. S. Bartlett and David Stirling, A Short History of the Polymerase Chain Reaction, in PCR Protocols, J.M.S. Bartlett and D. Stirling, Editors. 2003, Humana Press: Totowa, NJ. p. 3-6.
[4] B. J. Callahan, P. J. McMurdie, M. J. Rosen, A. W. Han, A. J. Johnson, and S. P. Holmes. DADA2: High-resolution sample inference from Illumina amplicon data, Nature methods, vol. 13, no. 7, pp. 581-583, 2016.
[5] Amnon Amir, Daniel McDonald, Jose A. Navas-Molina, Evguenia Kopylova, James T. Morton, Zhenjiang Zech Xu, Eric P. Kightley, Luke R. Thompson, Embriette R. Hyde, Antonio Gonzalez, and Rob Knight. Deblur Rapidly Resolves Single-Nucleotide Community Sequence Patterns, mSystems, vol. 2, no. 2, pp. e00191-00116, 2017.
[6] Jasmine Chong, Peng Liu, Guangyan Zhou, and Jianguo Xia. Using MicrobiomeAnalyst for comprehensive statistical, functional, and meta-analysis of microbiome data, Nature Protocols, vol. 15, no. 3, pp. 799-821, 2020.
[7] Achal Dhariwal, Jasmine Chong, Salam Habib, Irah L. King, Luis B. Agellon, and Jianguo Xia. MicrobiomeAnalyst: a web-based tool for comprehensive statistical, visual and meta-analysis of microbiome data, Nucleic acids research, vol. 45, no. W1, pp. W180-W188, 2017.
[8] Antonio Gonzalez, Jose A. Navas-Molina, Tomasz Kosciolek, Daniel McDonald, Yoshiki Vázquez-Baeza, Gail Ackermann, Jeff DeReus, Stefan Janssen, Austin D. Swafford, Stephanie B. Orchanian, Jon G. Sanders, Joshua Shorenstein, Hannes Holste, Semar Petrus, Adam Robbins-Pianka, Colin J. Brislawn, Mingxun Wang, Jai Ram Rideout, Evan Bolyen, Matthew Dillon, J. Gregory Caporaso, Pieter C. Dorrestein, and Rob Knight. Qiita: rapid, web-enabled microbiome meta-analysis, Nature methods, vol. 15, no. 10, pp. 796-798, 2018.
[9] Evan Bolyen, Jai Ram Rideout, Matthew R. Dillon, Nicholas A. Bokulich, Christian C. Abnet, Gabriel A. Al-Ghalith, Harriet Alexander, Eric J. Alm, Manimozhiyan Arumugam, Francesco Asnicar, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2, Nature Biotechnology, vol. 37, no. 8, pp. 852-857, 2019.
[10] Nick Weber, David Liou, Jennifer Dommer, Philip MacMenamin, Mariam Quiñones, Ian Misner, Andrew J Oler, Joe Wan, Lewis Kim, Meghan Coakley McCarthy, Samuel Ezeji, Karlynn Noble, and Darrell E Hurt. Nephele: a cloud platform for simplified, standardized and reproducible microbiome data analysis, Bioinformatics, vol. 34, no. 8, pp. 1411-1413, 2017.
[11] Gavin M. Douglas, Vincent J. Maffei, Jesse Zaneveld, Svetlana N. Yurgel, James R. Brown, Christopher M. Taylor, Curtis Huttenhower, and Morgan G. I. Langille. PICRUSt2: An improved and extensible approach for metagenome inference, bioRxiv, pp. 672295, 2019.
[12] R. Guerrero-Preston, F. Godoy-Vitorino, A. Jedlicka, A. Rodriguez-Hilario, H. Gonzalez, J. Bondy, F. Lawson, O. Folawiyo, C. Michailidi, A. Dziedzic, R. Thangavel, T. Hadar, M. G. Noordhuis, W. Westra, W. Koch, and D. Sidransky. 16S rRNA amplicon sequencing identifies microbiota associated with oral cancer, human papilloma virus infection and surgical treatment, Oncotarget, vol. 7, no. 32, pp. 51320-51334, 2016.
[13] D. Bornigen, B. Ren, R. Pickard, J. Li, E. Ozer, E. M. Hartmann, W. Xiao, T. Tickle, J. Rider, D. Gevers, E. A. Franzosa, M. E. Davey, M. L. Gillison, and C. Huttenhower. Alterations in oral bacterial communities are associated with risk factors for oral and oropharyngeal cancer, Scientific reports, vol. 7, no. 1, pp. 17686, 2017.
[14] R. B. Hayes, J. Ahn, X. Fan, B. A. Peters, Y. Ma, L. Yang, I. Agalliu, R. D. Burk, I. Ganly, M. P. Purdue, N. D. Freedman, S. M. Gapstur, and Z. Pei. Association of Oral Microbiome With Risk for Incident Head and Neck Squamous Cell Cancer, JAMA oncology, vol. 4, no. 3, pp. 358-365, 2018.
[15] W. H. Lee, H. M. Chen, S. F. Yang, C. Liang, C. Y. Peng, F. M. Lin, L. L. Tsai, B. C. Wu, C. H. Hsin, C. Y. Chuang, T. Yang, T. L. Yang, S. Y. Ho, W. L. Chen, K. C. Ueng, H. D. Huang, C. N. Huang, and Y. J. Jong. Bacterial alterations in salivary microbiota and their association in oral cancer, Scientific reports, vol. 7, no. 1, pp. 16540, 2017.
[16] B. Y. Hernandez, X. Zhu, M. T. Goodman, R. Gatewood, P. Mendiola, K. Quinata, and Y. C. Paulino. Betel nut chewing, oral premalignant lesions, and the oral microbiome, PloS one, vol. 12, no. 2, pp. e0172196, 2017.
[17] B. L. Schmidt, J. Kuczynski, A. Bhattacharya, B. Huey, P. M. Corby, E. L. Queiroz, K. Nightingale, A. R. Kerr, M. D. DeLacure, R. Veeramachaneni, A. B. Olshen, and D. G. Albertson. Changes in abundance of oral microbiota associated with oral cancer, PloS one, vol. 9, no. 6, pp. e98741, 2014.
[18] K. Hashimoto, D. Shimizu, S. Hirabayashi, S. Ueda, S. Miyabe, I. Oh-Iwa, T. Nagao, K. Shimozato, and S. Nomoto. Changes in oral microbial profiles associated with oral squamous cell carcinoma vs leukoplakia, Journal of investigative and clinical dentistry, vol. 10, no. 4, pp. e12445, 2019.
[19] X. Hu, Q. Zhang, H. Hua, and F. Chen. Changes in the salivary microbiota of oral leukoplakia and oral cancer, Oral oncology, vol. 56, pp. e6-8, 2016.
[20] D. Gopinath and R. K. Menon. Comments on "Compositional and functional variations of oral microbiota associated with the mutational changes in oral cancer" by Yang et al, Oral oncology, vol. 78, pp. 216-217, 2018.
[21] Z. Zhang, J. Yang, Q. Feng, B. Chen, M. Li, C. Liang, M. Li, Z. Li, Q. Xu, L. Zhang, and W. Chen. Compositional and Functional Analysis of the Microbiome in Tissue and Saliva of Oral Squamous Cell Carcinoma, Frontiers in microbiology, vol. 10, pp. 1439, 2019.
[22] R. Guerrero-Preston, J. R. White, F. Godoy-Vitorino, A. Rodriguez-Hilario, K. Navarro, H. Gonzalez, C. Michailidi, A. Jedlicka, S. Canapp, J. Bondy, A. Dziedzic, B. Mora-Lagos, G. Rivera-Alvarez, C. Ili-Gangas, P. Brebi-Mieville, W. Westra, W. Koch, H. Kang, L. Marchionni, Y. Kim, and D. Sidransky. High-resolution microbiome profiling uncovers Fusobacterium nucleatum, Lactobacillus gasseri/johnsonii, and Lactobacillus vaginalis associated to oral and oropharyngeal cancer in saliva from HPV positive and HPV negative patients treated with surgery and chemo-radiation, Oncotarget, vol. 8, no. 67, pp. 110931-110948, 2017.
[23] M. Perera, N. N. Al-Hebshi, I. Perera, D. Ipe, G. C. Ulett, D. J. Speicher, T. Chen, and N. W. Johnson. Inflammatory Bacteriome and Oral Squamous Cell Carcinoma, Journal of dental research, vol. 97, no. 6, pp. 725-732, 2018.
[24] N. N. Al-Hebshi, A. T. Nasher, M. Y. Maryoud, H. E. Homeida, T. Chen, A. M. Idris, and N. W. Johnson. Inflammatory bacteriome featuring Fusobacterium nucleatum and Pseudomonas aeruginosa identified in association with oral squamous cell carcinoma, Scientific reports, vol. 7, no. 1, pp. 1834, 2017.
[25] J. M. Shin, T. Luo, P. Kamarajan, J. C. Fenno, A. H. Rickard, and Y. L. Kapila. Microbial Communities Associated with Primary and Metastatic Head and Neck Squamous Cell Carcinoma - A High Fusobacterial and Low Streptococcal Signature, Scientific reports, vol. 7, no. 1, pp. 9934, 2017.
[26] A. Amer, S. Galvin, C. M. Healy, and G. P. Moran. The Microbiome of Potentially Malignant Oral Leukoplakia Exhibits Enrichment for Fusobacterium, Leptotrichia, Campylobacter, and Rothia Species, Frontiers in microbiology, vol. 8, pp. 2391, 2017.
[27] H. Wang, P. Funchain, G. Bebek, J. Altemus, H. Zhang, F. Niazi, C. Peterson, W. T. Lee, B. B. Burkey, and C. Eng. Microbiomic differences in tumor and paired-normal tissue in head and neck squamous cell carcinomas, Genome medicine, vol. 9, no. 1, pp. 14, 2017.
[28] C. Y. Yang, Y. M. Yeh, H. Y. Yu, C. Y. Chin, C. W. Hsu, H. Liu, P. J. Huang, S. N. Hu, C. T. Liao, K. P. Chang, and Y. L. Chang. Oral Microbiota Community Dynamics Associated With Oral Squamous Cell Carcinoma Staging, Frontiers in microbiology, vol. 9, pp. 862, 2018.
[29] Y. Lim, N. Fukuma, M. Totsika, L. Kenny, M. Morrison, and C. Punyadeera. The Performance of an Oral Microbiome Biomarker Panel in Predicting Oral Cavity and Oropharyngeal Cancers, Frontiers in cellular and infection microbiology, vol. 8, pp. 267, 2018.
[30] I. Ganly, L. Yang, R. A. Giese, Y. Hao, C. W. Nossa, L. G. T. Morris, M. Rosenthal, J. Migliacci, D. Kelly, W. Tseng, J. Hu, H. Li, S. Brown, and Z. Pei. Periodontal pathogens are a risk factor of oral cavity squamous cell carcinoma, independent of tobacco and alcohol and human papillomavirus, International journal of cancer, vol. 145, no. 3, pp. 775-784, 2019.
[31] N. N. Al-Hebshi, A. T. Nasher, A. M. Idris, and T. Chen. Robust species taxonomy assignment algorithm for 16S rRNA NGS reads: application to oral carcinoma samples, Journal of oral microbiology, vol. 7, pp. 28934, 2015.
[32] A. Wolf, C. Moissl-Eichinger, A. Perras, K. Koskinen, P. V. Tomazic, and D. Thurnher. The salivary microbiome as an indicator of carcinogenesis in patients with oropharyngeal squamous cell carcinoma: A pilot study, Scientific reports, vol. 7, no. 1, pp. 5867, 2017.
[33] H. Zhao, M. Chu, Z. Huang, X. Yang, S. Ran, B. Hu, C. Zhang, and J. Liang. Variations in oral microbiota associated with oral cancer, Scientific reports, vol. 7, no. 1, pp. 11773, 2017.
[34] NCBI. SRA-Toolkit. Available from: https://github.com/ncbi/sra-tools/wiki.
[35] Peter J. A. Cock, Christopher J. Fields, Naohisa Goto, Michael L. Heuer, and Peter M. Rice. The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants, Nucleic acids research, vol. 38, no. 6, pp. 1767-1771, 2009.
[36] Nicola Segata, Jacques Izard, Levi Waldron, Dirk Gevers, Larisa Miropolsky, Wendy S. Garrett, and Curtis Huttenhower. Metagenomic biomarker discovery and explanation, Genome Biology, vol. 12, no. 6, pp. R60, 2011.
[37] R. H. Whittaker. Vegetation of the Siskiyou Mountains, Oregon and California, Ecological Monographs, vol. 30, no. 3, pp. 279-338, 1960.
[38] R. H. Whittaker. EVOLUTION AND MEASUREMENT OF SPECIES DIVERSITY, TAXON, vol. 21, no. 2-3, pp. 213-251, 1972.
[39] 謝維馨/ 有勁生物科技. 學生t檢定. Available from: https://yourgene.pixnet.net/blog/post/117254074-%E5%AD%B8%E7%94%9Ft%E6%AA%A2%E5%AE%9A-(student%E2%80%99s-t-test).
[40] Wiki. Wilcoxon rank-sum test. Available from: https://en.wikipedia.org/wiki/Mann%E2%80%93Whitney_U_test.
[41] Wiki. K-S test. Available from: https://en.wikipedia.org/wiki/Kolmogorov%E2%80%93Smirnov_test.
[42] Wiki. Kruskal–Wallis test Available from: https://en.wikipedia.org/wiki/Kruskal%E2%80%93Wallis_one-way_analysis_of_variance.
[43] S. Mandal, W. Van Treuren, R. A. White, M. Eggesbø, R. Knight, and S. D. Peddada. Analysis of composition of microbiomes: a novel method for studying microbial composition, Microbial ecology in health and disease, vol. 26, pp. 27663, 2015.
[44] Jonathan Friedman and Eric J. Alm. Inferring Correlation Networks from Genomic Survey Data, PLOS Computational Biology, vol. 8, no. 9, pp. e1002687, 2012.
[45] Stilianos Louca and Michael Doebeli. Efficient comparative phylogenetics on large trees, Bioinformatics, vol. 34, no. 6, pp. 1053-1055, 2017.
[46] Yuzhen Ye and Thomas G. Doak. A Parsimony Approach to Biological Pathway Reconstruction/Inference for Genomes and Metagenomes, PLOS Computational Biology, vol. 5, no. 8, pp. e1000465, 2009.
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