高通量篩選技術研討會

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(2) 高通量篩選技術研討會 2009 年 10 月 1 - 2 日. 系統生物學是一個試圖整合不同層次信息以理解生物系統如何行使功能的學術領域。通過研究模式生物系統各不同部分之間的相互關係和相互作用（例如，與細胞信號傳導，代謝通路，細胞器，細胞，生理系統與生物等相關的基因和蛋白網路），期望最終能夠建立整個系統的可理解模型。系統生物學是將 DNA、RNA、蛋白質以及三者彼此之間的交互作用等資訊加以整合，並運用這些資料去建立出數學計量模型，以期能掌握所有生物基因與組織間的關係及運作。因此系統生物學的研究過程是先取得一個生物、組織或細胞系統，辨識出各種內部因素，在獲得 DNA、RNA 及蛋白質相互作用及資訊網路方面整合所獲得的資訊，然後開發出能描述系統結構和行為的數學模型，最後可以藉由此一個模型系統，使這個系統可以自動執行所需的功能。因此系統生物學是結合許多不同學科的領域，透過彼此相互的網狀合作，針對一個生物現象所進行的研究。. 系統生物學與以往的實驗生物學不同的是它要研究所有的基因、所有的蛋白質、組成份間所有的關係如基因網路和蛋白質和蛋白質間交互作用；並利用計算生物學及生物資訊學來定量描述和預測生物功能、表型和行為。基因體分析等研究為我們累積了巨量的數據，而生物資訊學可協助我們利用這些數據加速實驗的預測與模組建立，也因此有機會從整體的、合成的角度檢視生物學，而建立所謂的系統生物學。未來的生物學會由傳統的描述性科學，轉變成一種可定量、可分析及具有預測性的生物學，所以需要借助數理工程領域的研究方法，如此才能使得生物學如物理科學般有數學模型的理論基礎，這些理論是由有定量化的實驗證據而建立的，不但可用來解釋實驗觀察到的生物現象，而且可進一步預測其它可能的生物反應。因此，系統生物學在未來生物科技的發展是深具潛力且重要的。系統生物學透過使用高通量技術來測定某物種在給定條件干涉下基因組和蛋白質組的變化。研究基因組的高通量技術包括高速定序技術，mRNA 變化的生物晶片技術。高通量蛋白質組學方法包括質譜，該技術用於鑒定蛋白質，檢測蛋白修飾和量化蛋白質表達水平。因此，高通量篩選技術變成產成大量訊息必備的分析方法與必要手段。此次舉辦的跨領域交流之高通量篩選技術研討會，其目的在於對各式新興的高通量篩選技術進行新知交流，拉進生物資訊學者與生物學者間的交流與合作，並吸引生科及電資學院學生進入系統生物學領域。此外，本學術研討會將邀請友校與業界的相關學者一同參與，除了可以交流彼此的研究心得，也可藉由本研討會加強彼此的合作關係，建立更完善的跨校合作系統。. 辛致煒成功大學醫學院微免所 2009.09.28.

(3) 高通量篩選技術研討會 2009 in NCKU. 2009.10.01. ii.

(4) 高通量篩選技術研討會 2009 in NCKU. 2009.10.02. 主辦單位：國立成功大學醫學院微免所協辦單位：教育部、國立成功大學舉辦時間：中華民國九十八年十月一、二日舉辦地點：國立成功大學總圖書館地下一樓國際會議廳贊助廠商：均泰生物科技有限公司、美商應用生命系統股份有限公司. 、金萬林企業股份有限公司計畫主持人：辛致煒計畫同主持人：張翠砡封面設計：江嘉琪. iii.

(5) 演講摘要.

(6) Comparison between the ribo-minus RNA-sequencing and polyA-selected RNA-sequencing. Songnian Hu 胡松年教授 Beijing Institute of Genomics, Chinese Academy of Sciences, No. 7 Beitucheng West Road, Chaoyang, Beijing, China 中科院北京基因組研究所，浙江大學 Tel: 86-10-82995362; Fax: 86-10-82995362 e-mail: [email protected].. Abstract. To determining which method, the ribo-minus RNAsequencing (rmRNA-seq) or polyA-selected RNA-sequencing (mRNA-seq), is more suitable for the whole transcriptome analysis, we applied both methods to sequence the whole transcriptome of mouse cerebrum, and performed comparative analysis between both datasets. Using rmRNA-seq and mRNA-seq, we separately generated 140 and 93 million mappable reads of 35 bases in length, and 25% and 40% of the sequence reads can be mapped to the unique genomic region. First, we found higher proportion (47% and 53%) of uniquely mappable rmRNA-seq reads are in the intergenic and intronic regions, respectively, compared to 60% and 40% of the mRNA-seq reads, which determined that more intergenic and intronic transcribed regions can be identified with rmRNA-seq. Further comparative analysis suggested the rmRNAseq method can detect more transcripts mainly from the intergenic and intronic regions, including some well-known ncRNAs, such as mRNA-like ncRNAs, snoRNA and snRNA, and newly-identified transcripts supported by RT-PCR result. In addition, we observed 1.

(7) 高通量篩選技術研討會 2009 in NCKU. the reads from rmRNA-seq show a more uniform distribution across genes compared to those from mRNA-seq, considered that rmRNA-seq method can exclude the effects of the truncation of the 5’ portion of RNA isolated by oligo-dT. Above all, these results provided novel insight that rmRNA-seq could be a better way to more accurately and completely study the eukaryotic transcriptome.. !. !. 2.

(8) A hallmark of DNA damage: Hydroxyurea (HU)-induced global transcriptional suppression. Songnian Hu 胡松年教授 Beijing Institute of Genomics, Chinese Academy of Sciences, No. 7 Beitucheng West Road, Chaoyang, Beijing, China 中科院北京基因組研究所，浙江大學 Tel: 86-10-82995362; Fax: 86-10-82995362 e-mail: [email protected].. Abstract ! Combining high-throughput sequencing and microarray data, we show that the global transcriptional activity is suppressed as mouse ES cells are exposed to hydroxyurea (HU)-induced DNA damage. This transcriptional suppression is global regardless if the transcripts are exonic, intronic, and intergenic, leading to downregulation of most coding and non-coding genes in the HU-treated cells and altering multiple key cellular pathways. Our result is consistent with the cell-level observation that ubiquitylation- and proteasome-mediated degradation of RNA polymerase II, triggered by DNA damages generally leads to the inhibition of transcription. We propose a global transcriptional suppression as a hallmark of cellular DNA damage and postulate the mechanisms how HU may act on cell cycle, cell apoptosis, and other related DNA repair pathways. Our result provides novel implications for the understanding of how the cells respond to chemotherapeutic compounds such as HU. 3.

(9) A systems biology approach reveals the evolution of pathways deregulated in the hepatocellular carcinogenesis and the critical genes. Chiou-Hwa Yuh 喻秋華教授 Division of Molecular and Genomic Medicine, National Health Research Institutes, College of Life Science and Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Department of Biological Science & Technology, National Chiao Tung University 國家衛生研究院竹南院區，苗栗縣竹南鎮科研路 35 號 Tel:(037) 246-166 ext. 35338 e-mail: [email protected]. Abstract. Hepatocarcinogenesis is a slow and multistep process. An accumulation of genomic changes involved in different regulatory pathways is believed to be causal in liver cancer. In the present study, we systematically delineated the molecular events during different stages of hepatocarcinogenesis using the HBx transgenic mouse model. We analyzed gene expression levels that differed at least two-fold between HBx transgenic and wild-type mouse livers. Statistical approaches were used to identify genes that displayed an increasing or decreasing trend throughout hepatocarcinogenesis, as they may be associated with disease progression. We identified the pathways activated or repressed during tumor formation, describing in detail the interrelationships responsible for hepatocarcinogenesis. Furthermore, using sub-network enrichment analysis in PathwayStudio 6.0, we identified five common regulators of multiple genes that are differentially over-expressed at 14 4.

(10) 高通量篩選技術研討會 2009 in NCKU. versus 12 months. Three of these (edn1, bmp7 and bmp4) are upregulated at the 12 months stage. Two (spib and src) are upregulated at the 14 months stage. They may promote carcinogenesis via up-regulation of pre-cancer oncogenes. Expression of the common regulators and their target genes was verified by real time RT-PCR. The systematic approaches used in this project have helped to elucidate the basis of HCC carcinogenesis. The results will also help to assess the possibility of utilizing the biomarkers identified as molecular targets for treating hepatocellular carcinoma. The microarray data presented in this study have been approved by Gene Expression Omnibus (Series accession number GSE15251, along with microarray experimental design, data collection and analyses). All the results presented in this study are freely available for download and query at http://www.HBx-LiverCancer.org/.. !. !. 5.

(11) Comparative analysis of co-expressed protein interaction networks. Hsuan-Cheng Huang 黃宣誠教授 Institute of Biomedical Informatics, National Yang-Ming University, Taipei, Taiwan 國立陽明大學生物醫學資訊所，台北市北投區石牌立農街二段 155 號 Tel:(02)28267000 ext 6169 e-mail: [email protected]. Abstract. As molecular networks represent the backbone of molecular activity within cells, integrative analysis of transcriptomic profiles in the context of protein interaction networks provides opportunities for understanding the molecular mechanism of diseases. While protein-protein interaction data constitute static network maps, integration of condition-specific co-expression information provides clues to the dynamic features of the networks. Here, we present a network-based comparative analysis that integrates gene expression profiles with protein-protein interaction and biological function annotations to elucidate heart failure related molecular modules. The revealed hub genes and molecular modules may be used as potential disease markers and provide new directions for heart failure therapy.. 6.

(12) Data-driven cancer gene expression network analysis. Jung-Hsien Chiang 蔣榮先教授 Institute of Medical Informatics, Dept. of Computer Science and Information Engineering, National Cheng Kung University, Taiwan 國立成功大學資訊工程學系，台南市大學路 1 號 Tel:(06)275-7575 ext 62534 e-mail: [email protected]. Abstract. The aim of this speech is to introduce an integrated systems biology infrastructure for cancer genes screen and annotation based on dry-lab approach. In specific, we establish a multifunctional bioinformatics platform for effectively finding gene-pair relations, clustering gene expression profiles, and mining target gene modules from vast amount of information available in both microarray data and biomedical text for cancer study. This infrastructure allows us to rapidly discover biological functions of target genes in the prostate cancer related research, and the identified candidate genes can be further studied in terms of biological functions and molecular mechanisms by wet lab experiments to confirm the prediction from in silico approach. We also present a framework for discovering and visualizing functional modules from protein-protein interactions through network clustering and Gene Ontology classifications. The integrated system implements the proposed framework such that medical researchers can gain valuable insights into the inter-relations of their genes of interests through auto-generated interactive cellular diagrams for module 7.

(13) 高通量篩選技術研討會 2009 in NCKU. groups along with the correlation matrices to associated pathways and annotations.. !. !. 8.

(14) Evolution and biological role of microRNA in protist. Petrus Tang 鄧致剛教授 Molecular Regulation & Bioinformatics Laboratory, Chang Gung University, Taiwan, Division of Microbiology, Graduate Institute of Biomedical Sciences, Chang Gung University, Taiwan, Molecular Medicine Research Center, Chang Gung University, Taiwan. 長庚大學公共衛生暨寄生蟲學科，桃園縣龜山鄉文化一路 259 號 Tel:(03)211-8800 ext 5136 e-mail:[email protected]. Abstract MicroRNAs (miRNAs) are a class of small noncoding RNAs that can suppress protein translation through complementary binding to target mRNAs. A large number of miRNAs have been characterized from almost all eukaryotic species since the discovery of the first two miRNAs, lin-4 and let-7 in the 1990s. However, very little is known about the regulation of miRNA in protist. By using microRNA serial analysis of gene expression (miRAGE), we identified nine candidate miRNAs in the parasitic protist Trichomonas vaginalis (Tv-miR-001 to 009). None of these protist miRNAs showed significant sequence homology with known miRNAs deposited in the miRBase. To explore the evolutionary origins of metazoan miRNAs, we sequenced three small RNA libraries by using the Illumina Genome Analyzer platform. A total of 652,088 tags which composed of 1,828,1492 reads were subject to a DeepSequencing Small RNA Analysis Pipeline (DSAP) to remove small 9.

(15) 高通量篩選技術研討會 2009 in NCKU. non-coding RNA populations other then miRNA, to identify tags with sequence homology with known miRNAs. Only 39 T. vaginalis matched to known miRNAs. Among these newly identified protist miRNAs, some miRNAs are previously considered as plant, arthropods or muscle specific (miR-1). This raised an interesting question on the evolution of miRNAs. It appears that T. vaginalis is the ancestor of many organism or tissue specific miRNAs. We also used a novel approach to identify miRNA targets in the protist T. vaginalis by comparing the levels of differentially expressed proteins and genes in the trophozoite and amoeboid stages. We observed that the T. vaginalis malate dehydrogenase (Tv_MDH) gene was up-regulated 20-fold in the amoeboid stage, but the protein level was reduced by 4.5-fold. Bioinformatics analysis revealed that the Tv_MDH mRNA contains putative target sites of the miR-1 family. We showed that the expression level of endogenous tvamiR-1 in the amoeboid stage was 50-fold higher than in the trophozoite stage. Transfection of trophozoites with tva-miR-1 mimics reduced Tv_MDH protein expression by 60%. We concluded that Tv_MDH is negatively regulated by tva-miR-1. Based on the experimental results of the present study, we provided solid evidence that protist possess an ancient miRNA regulating network.. !. !. 10.

(16) Gene ontology: Tool for the unification of biology. Yow-Ling Shiue 薛佑玲教授 Institute of Biomedical Science, National Sun Yat-sen University 國立中山大學生物醫學研究所，高雄市鼓山區蓮海路 70 號 Tel:(07)525-2000 ext 5818 e-mail:[email protected]. Abstract Genome projects across several hundreds of species have made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Indeed, almost all important biological pathways were first found in model organisms. Knowledge of the biological role of such shared proteins in model organisms can often be transferred to higher organisms, including the human. However, the speed of data accumulation is far higher than the knowledge accumulation. Recently, bioinformatics such as Gene Ontology (GO) provides a way to capture and represent biological knowledge in a computable form. The GO project is maintained by the GO consortium, aiming to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http//www.geneontoloy.org) have been established. So far, 11.

(17) 高通量篩選技術研討會 2009 in NCKU. GO terms are widely adopted in genomic as well as valueadded databases, across almost all species. In addition, using the GO for expression analysis; finding the biological process, molecular function, and subcellular component of novel proteins, are commonly applied.. !. !. 12.

(18) Large scale bioinformatic discovery of microRNAs in metazoan genomes and experimental validation. Wen-chang Lin 林文昌教授 Institute of Biomedical Sciences, Academia Sinica. 中央研究院生物醫學科學研究所，台北市南港區研究院路二段 128 號 Tel:(02)2789-9002 e-mail:[email protected]. Abstract MicroRNAs (miRNAs) are endogenous non-protein-coding RNAs of ~22 nucleotides. Since the initial discovery of miRNAs in Caenorhabditis elegans, thousands have been identified (computationally and/or experimentally) in many organisms, including mammals, invertebrates, insects, plants and viruses, which suggests that miRNA genes have been conserved during evolution and widely distributed among species. Here, we modified our previous pre-miRNA discovery pipelines to predict miRNAs in more than fifty metazoan genomes. Using the support vector machine as a classifier, we identified an additional 17,479 orthologous or paralogous pre-miRNAs, as well as their corresponding mature miRNAs, with 89.5% sensitivity and 97.4 % specificity. In order to perform large scale validation of prediction results, we selected rabbit (Oryctolagus cuniculus) and medaka (Oryzias latipes) for further small RNA sequencing validation with the SOLiD next generation sequencing platform. Originally, our pipeline 13.

(19) 高通量篩選技術研討會 2009 in NCKU. predicted 273 and 354 candidate pre-miRNAs in medaka and rabbit, respectively. After processing the SOLiD reads, we defined a matched candidate mature miRNA must be 100% identical to the SOLiD reads. Totally, 529,396 medaka reads are matched by medaka candidates; in rabbit, this value is 1,703,581. Besides, 79.9% (218 out of 273) medaka candidate pre-miRNAs have SOLiD reads matched their corresponding miRNAs; 59.3% (210 out of 354) rabbit candidate pre-miRNAs do so. Our results suggest that miRNA genes are widely distributed in many animal species, including Schmidtea, nematode, insect, urchin, sea squirt, and vertebrates. Different miRNA families have distinct distribution patterns among these species, which may provide insight into miRNA evolution and their functional significance in development and organogenesis.. !. !. 14.

(20) 誰需要生物晶片分析？ Who needs microarray analysis?. Bei-Chang Yang 楊倍昌教授 Department of Microbiology and Immunology, National Cheng Kung University 國立成功大學醫學院微生物及免疫學研究所，台南市大學路 1 號 Tel:(06)2353535 ext 5637 e-mail:[email protected]. Abstract 生物科學的研究五花八門，各個年代都有各自的流行主題。由資料的性質來看，近代生物科學可以簡單的標示出三項共同趨勢：「更快」、「更細」與「更多」。「更快」的意思是觀察資料能及時呈現生物反應。生物是個開放而且能夠和環境交互影響的動態系統，生命現象從未停滯不前。資料的截取的時間越短就越可以實際呈現快速變化的生命現象。要能達到及時呈現生命的變化的境界，必須儘量不干擾所觀測的生命系統。以往將生物殺死、固定而記錄單一生命表徵的實驗方式，所得到的結果只像是分離而單一的照片；而近代科學實驗所希望紀錄的是活生生的生物，是連續的生命活動，需要有及時觀察的電影記錄片效果。活體觀察是「更快」的要求之下不得不然的實驗條件。「細」則是空間的壓縮。生命現象是實存物的展現。實存物占有空間。新工具所量測的有效距離越短，描述物的結構越細緻，生命物的輪廓就越清晰。最近科學期刊雜誌上所發表文章的圖像的品質越來越高，新的光學儀器越來越昂貴、解析力越來越好，這些都能反映出近代科學對追求「更細」的資料的取向。簡單的說，「更快」是時間的切割、「更細」是空間 15.

(21) 高通量篩選技術研討會 2009 in NCKU. 的切割。它們所努力的是對資訊品質的改變。這種方法學所處理的時空是化約論（Reductionism）對知識的態度。化約論者認為複雜的現象或系統都可以分解成各小部分而加以理解和描述，這是西方的科學哲學的傳統老辦法。法國哲學家笛卡爾（Rene Descartes， 1596-1650）在『方法論』中建議1.懷疑一切，2.將複雜的問題拆散成較簡單的小問題，3.先由容易的小問題下手，4.整併結果、綜合討論。科學研究要求「更快」、「更細」的過程不斷的將時空割裂成更小的片段，正好符合笛卡爾的方法學中所建議拆解式的研究取向。「更多」所注重的是資訊的數量。要求「更多」資訊可以有幾種的方法：長時間的資料累積、鼓勵更多研究者投入、透過高通量式 (high-throughput) 的檢測方式等等都算是。以高通量式的檢測，快速取得鉅額的資料則是近來最流行的策略。為了得到大量的資料，在方法上會儘可能的操作自動化、材料迷你化、資訊管理電腦化。高通量式的檢驗動則成千上萬，材料及試劑的製備大多非常單調，使用人力操作枯燥而反復的工作非常如容易出錯也不符合成本。因此，由電腦程式操控的自動化機械是最佳的選擇。為了取得大量的資料，試劑和生物檢體的需求也大，相對的實驗耗材成本會非大。將檢驗反應迷你化不只是成本考量，也是有效的利用生物檢體的對策。至於高通量式檢驗的成果分析更是非借助電腦運算不可。高通量式的檢驗所讓人困擾的缺失絕對不是資料不足，反而是多到像漫天飛舞的蝗蟲，讓人分不清楚那一筆資料才富有生物意義。生物晶片則是高通量式檢測最典型的代表，它完全符合操作自動化、材料迷你化、資訊管理電腦化的技術特徵。生物科學的分析中要求「更多」的本質是什麼？「更快」「更細」所對付的是物質世界最根本的時空結構，它們是認知對於事件的品質的自我要求。就認識論的內涵來看，「更多」所思考的範疇不在時空的問題上。「更多」本身與自我淬鍊無關，它是由於生命現象的特殊性而不得不調整的措施，是一種附屬的導出量。「更多」的概念隨附 (supervenience) 在生物的多型性 (polymorphism; diversity) 和網絡式的多基因控制單一生物表徵 (multiple genes-regulated phenotype)。在實際的觀察經驗下，生物與非生物的物性有極大的不同。非生物如礦物之類的物質的同質性很高，它們所呈現的物理量也相當一致，個體之間的差異不大。但是生物則不同。生物的基因多型性 (multiple alleles) 是常態，同種生物的 16.

(22) 高通量篩選技術研討會 2009 in NCKU. 個體之間都有些許差異。生命現象的表現多是鐘型的機率分佈 (bell-shaped distribution)，最高觀察值與最低值的差距大。就以簡化的基因多型性來說，同一基因可以因為少數核酸序列的更動而改變表現效率、穩定度、對應蛋白質的酵素功能。單一基因到單一性狀是最簡單的遺傳控制模式。撇開環境的因素不談，身高、膚色、生命周期、情緒甚至是遺傳性疾病多是由多個基因所控制。一般所稱的正常典型，只是用來指稱當族群當中可觀察的頻率最高的類型而已。此外網絡式的多基因控制則更複雜。由基因序列到基因/ 蛋白質的表現除了必需經過轉錄、轉譯、傳送之外，基因之間還有不同訊息之間的交互影響 (cross-talk)，讓不同的基因可以組成群集。甚至由細胞層次進到可觀察的生物特性還有層層疊疊的調控，複雜的不得了。這些生命表徵的差異只能透過統計分析的方式來處理。透過分析大量的檢體來擴大母數而加強平均質的說服力。目前人的知識無法處理那麼複雜的系統，勉強只能以「數大為美」的方式來趨近真相。因此在生物科學家的潛意識裡、資料總是要越多越好，用來安慰自己免得掛一漏萬。隨附在生物多型性的「更多」的傾向，成了實驗設計上不需論證的反射動作。在思維上，附屬在生物性而要求「更多」與處理時空結構的「更快」、「更細」也不同。在科學文章中，陳述「更快」、「更細」的實驗需要先有完整的因果論述。就時間而言，研究者心裡在實驗設計當下必然有特定的理由，有專一的標的，需要在某個時段，及時的觀察某個生物反應的變化。分析生物的空間結構要求「更細」也要先交代清楚的邏輯。否則，就算是面對非常細緻的細胞結構圖，沒有特定的目標，人還是會視而不見。相對的，高通量式的檢測策略大多不必有嚴緊的因果論述。就我的觀察，高通量式的檢測結果大多是倒果為因，直接作為因果論述的起點。它很像是一種胡亂撒網補魚法，先在混水中抓到了魚之後再來編排故事。這類文章的寫法一般都會先說「使用了基因晶片(或是其它高通量式的檢測方法)發現了某現象很有趣(有趣不有趣大多是主觀的說詞)，因此接著分析該現象，而結果又如何如何….」。研究者的功力大多呈現在他的財力夠不夠雄厚、編故事的功夫高不高明，運氣是否好到能撈到珍貴的魚。偏重「更快」、「更細」的實驗強調無中生有的想像力、勤勉的文獻閱讀以及嚴緊的邏輯分析，執行實驗之前的規劃多半小心奕奕。此外這兩類實驗對於精密機器的依賴性很高，因此 17.

(23) 高通量篩選技術研討會 2009 in NCKU. 硬體投資相當重要。而高通量式的實驗則不怕浪費。解讀「鉅量資料」則需要高明的詮釋能力以及強大的電腦運算能力，所以投資重點應該注重在強化軟體的分析功能。對現代科學家來說，如果暫時沒有什麼美妙的研究題材，而研究經費還算充裕，那麼做些晶片分析、掃瞄蛋白質體、透過高通量的技術篩選大批的天然/合成藥物都會是不錯的選擇。. 18.

(24) From 1 to many - 高通量表達實驗策略. Jyh-wei Shin 辛致煒教授 Department of Microbiology and Immunology, National Cheng Kung University 國立成功大學醫學院微生物及免疫學研究所，台南市大學路 1 號 Tel:(06)2353535 ext 5586 e-mail: [email protected]. Abstract 基因差異表達 (gene differential displaying) 高通量分析是分子生物學有力的分析工具。高通量篩選技術 (high throughput screening) 近年來發展迅速，應用越來越廣泛。該方法集計算機控制、自動化操作、高靈敏度檢測、數據結果的自動收集和處理於一體。適用於以生物標記分子或細胞功能為基礎的分析實驗。隨著多種生物基因組全長序列的公布，研究重點逐漸從基因體學轉移到蛋白質組學上來，各項研究也愈發對不同細胞或不同狀態下基因表達差異產生興趣。近年來由於系統生物學技術的發展，從定序，基因表達，蛋白質表現與交互作用，表達網絡到生物影像都建立起高通量的實驗技術。由於這些技術都有別以往的差異表達實驗，在實驗上要有更多面向的思維。本次研討會將以膀胱癌作為研究題材，利用高通量技術在四大體學下進行分析，期找出有關膀胱癌的生物標記。. 19.

(25) Enable revolutionary genomics research by illumine now generation sequencing technology. 彭英哲 Genetech Biotech Co., tLd. 均泰生物科技有限公司，台北市松山區南京東路三段 338 巷 3 號 7 樓 Tel:(02) 2796-5108 e-mail:[email protected]. Abstract Illumine 的 Genome Analyzer IIx 是設計用來針對現今重要的基因遺傳研究來提供有效的分析工具，包括全基因體的再定序 (Whole genome resequencing)，特定區域再定序 (candidate region sequencing), 全基因體基因表現 (genome wide expression profiling), 轉錄體表現分析 (Transcriptome analysis)，微小RNA 分析 (Small RNA identification and quantification), ChIP 定序分析。目前許多的研究都可以利用一套系統來達成。這套系統可以提供高速有效的分析且非常經濟的費用就可以來改善現有技術的一些問題與困難, 或是進行以往無法完成的研究分析，利用這套系統，搭配現有的一些分子生物學的技術，即可完成多種的研究分析，有如在實驗室中建立一個小型的基因體中心一般。 Illumina 利用特有的 Paired-end 定序技術配合 Mate-Pair 技術，可使用於片段長度 200 bp - 5 kb 的基因庫構築，可提供平均且彈性的定序覆蓋面。每條 DNA 可依應用選擇定序 18 - 75 個鹼基, 並可搭配 Pair-end 進行雙端定序，(75 x 75bp)，每次可產生 25 Gb 精確且高品質的序列資料。. 新一代突破性的定序平台是基於大規模平行測序技術，使用專利可逆終止的測序化學方法。這種新穎的兼具成本效益和準確性的下一代測序技術測序，提供生物學研究一個高度可靠、準確、可擴展 20.

(26) 高通量篩選技術研討會 2009 in NCKU. 之分析工具。. !. !. 21.

(27) The Roche genome sequencer FLX titanium system Most update technology and applications. Wendy Huang 黃玟寧 Roche Applied Science, Taiwan 羅氏醫學儀器股份有限公司應用科學部，台北市中山區民生東路三段 2 號五樓 Tel:(02)21836688 ext 614 e-mail:[email protected]. Abstract The Genome Sequencer FLX System (GS FLX) is a nextgeneration DNA sequencing technology featuring a unique mix of long reads, exceptional accuracy, and ultra-high throughput. This very versatile next-generation sequencing technology, supports many high profile studies in many application categories. GS FLX users have pursued innovative research in de novo sequencing, resequencing of whole genomes and target DNA regions, metagenomics, RNA and trancriptome analysis. 454 Sequencing is a powerful tool for human genetics research like re-sequencing the genome of an individual human, the complete human exome and targeted genomic regions using the NimbleGen sequence capture process, detection of low frequency somatic mutations linked to cancer and analysis of genomic structural variations. Also sequencing of highly polymorphic regions like HIV genomes and the human HLA locus were addressed by utradeep amplicon sequencing. The FLX Titanium long reads of ~400bp combined with a new paired end protocol are widely used for de novo shotgun sequencing of complex eukaryotic organisms like plant genomes. 22.

(28) 高通量篩選技術研討會 2009 in NCKU. This presentation will provide a short overview about the 454 Sequencing technology, and will focus on all major areas of applications possible to address with the Genome Sequencer FLX system, from de-novo and transcriptome analysis to metagenomic sequencing. It will also provide information about the latest product developments, including the recent launch of Titanium kits for shotgun sequencing generating more than 500 Megabases per run (5 gigabases per week) at an average read length of ~400 bases on the FLX instrument.. !. !. 23.

(29) From Genome to Transcriptome- The Application of Next Generation Sequencing Technology with SOLiD. Kevin Wang 王簾讀 Applied Biosystems, the part of Life Technologies LLC 美商應用生命系統股份有限公司台灣分公司台北市中正區忠孝東路二段123號8樓 Tel:(02)23582838 ext 8602 e-mail:[email protected]. Abstract The SOLiD™ System is a highly accurate, massively parallel next-generation sequencing platform that supports a wide range of applications. The flexibility of two independent flow cells and multiplexing capability allow you to conduct multiple experiments in a single run. With unparalleled throughput and greater than 99.94% basecalling accuracy, the SOLiD™ System enables you to complete large-scale sequencing and tag experiments more cost effectively than previously possible.. The SOLiD System’s open slide format and flexible bead densities enable increases in throughput with protocol and chemistry optimizations. While competitive technologies are already near, or have achieved maximum throughput, the SOLiD System has room for throughput advancements utilizing the same platform.. With system accuracy greater than 99.94%, due to 2 base encoding, the SOLiD™ System distinguishes itself by providing data that is significantly more accurate than alternative nextgeneration platforms for variation detection. 2 Base encoding en24.

(30) 高通量篩選技術研討會 2009 in NCKU. ables unique error checking capability, providing higher confidence in each call. With the SOLiD System, scientists can focus on the biological significance of their results rather than sifting through poor quality data.. The SOLiD™ 3 System generates over 20 gigabases and 400M tags per run, which is more usable data than any other next-generation system available today. This level of throughput enables large scale resequencing and tag based experiments to be completed more cost effectively than ever before.. The independent flow cell configuration of the SOLID Analyzer enables you to run two completely independent experiments in a single run—essentially providing 2 instruments in one. The combination of multiple slide configuration and sample multiplexing capability enables you to analyze multiple samples cost effectively for a variety of applications.. !. !. 25.

(31) CLC bio - the world's leading bioinformatics solution provider!. Henry Wang 王昀珩 CLC bio Finlandsgade 10-12 Katrinebjerg, 8200 Aarhus N Denmark Kim Forest Enterprise., Ltd. 金萬林企業股份有限公司台北市信義區忠孝東路五段 510 號 15 樓 Tel:02-27260768. Abstract CLC bio is the world's leading bioinformatics solution provider. Next Generation Sequencing is a major focus area and CLC bio delivers the first and only comprehensive cross-platform analysis solution, which can analyze and visualize genomic, transcriptomic, and epigenomic data from all major platforms, Illumina’s Genome Analyzer, SOLiD by Applied Biosystems, 454 by Roche, and HeliScope by Helicos. Some of the key Next Gen Sequencing applications of CLC Genomics Workbench are: Genomics: * Whole genome re-sequencing and targeted re-sequencing of genomes of any size and type – from bacteria and vira to humans * de novo sequencing of an unlimited number of reads, for genomes up to 50 mega bases 26.

(32) 高通量篩選技術研討會 2009 in NCKU. * SNP detection, DIP detection, and identification of genomic rearrangements * Visualization and interactive graphical manipulation of results Transcriptomics * Digital Gene Expression based on RNA-Seq, including a wide range of downstream gene expression analyses * Discovery of novel transcripts/exons * Ability to work with Expression Arrays and RNA-seq results at the same time, enabling comparison of results * Interactive views of assemblies and derived gene expression data Epigenomics * Chromatin immunoprecipitation sequencing (ChIP-seq) analysis * Peak finding and peak refinement * Graph and table of background distribution and false discovery rate * Peak table and annotations. !. !. 27.

(33) Target enrichment technology significantly improves the cost- and process-efficiency to next-generation sequencing. Yi-Shing Lin 林怡杏 Welgene Biotech. Co., Ltd. 威健股份有限公司 Tel: 02-66160001 ext 22 e-mail:[email protected]. Abstract While next-generation sequencing has revolutionized the way genomes are sequenced, this technology possesses a fundamental weakness—the inability to easily target specific regions of a genome. To address this, Agilent has released the highly powerful SureSelect Target Enrichment System, developed in collaboration with the Broad Institute.1 This system uses an extremely efficient hybrid selection technique which significantly improves the cost- and process efficiency of the sequencing work flow, allowing for a larger number of samples per study. With sample input requirements at or below 3µg of genomic DNA, even the most precious of samples can be utilized for massively-parallel sequencing without risk of depletion. The system leverages well-known Agilent strengths: 1) proprietary SurePrint oligonucleotide synthesis of complex libraries consisting of ultra-long oligonucleotides greater than 100 bases in length; 2) custom library design fully integrated with eArray, Agilent’s on-line design tool; 28.

(34) 高通量篩選技術研討會 2009 in NCKU. and 3) quality manufacturing processes that ensure the greatest reliability and consistency. The system can also easily be incorporated into an automated environment, further increasing process efficiencies, while minimizing total sample costs.. !. !. 29.

(35) Advancing Discovery with High Density Functional Protein Microarrays. Timothy Wong 黃永亮 Market Development Manager, Pharma and CRO, Asia Pacific Invitrogen, part of Life Technologies 萊富生命科技股份有限公司 e-mail: [email protected] website: www.invitrogen.com/drugdiscovery. Abstract With the seminal work in Michael Snyder’s laboratory at Yale and Stuart Schreiber’s laboratory at Harvard few years ago, it was demonstrated that functional protein microarrays provide miniaturized high-throughput tools to elucidate biological pathways, screen biomarkers and engage in drug development. The generation of microarrays comprising whole proteomes of organisms will make significant contributions towards our understanding of disease pathways. The ProtoArray® Human Protein Microarray v5.0 is an advanced, high content, functional protein microarray that enables scanning of thousands of proteins for biochemical interactions in as little as one day. The new version ProtoArray comprises over 9,000 human proteins representing multiple gene families arrayed in duplicate on 76 x 26mm glass slide. During this seminar, various a p p l i c a t i o n s , s p e c i fi c a l l y, p ro t e i n - p ro t e i n i n t e r a c t i o n , kinase-substrate interaction, small molecule profiling and immune response biomarker profiling, with ProtoArray® microarray technology will be discussed. 30.

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(38) www.roche-applied-science.com Genome Sequencer FLX System. Introducing the GS FLX Titanium Reagents. Length Really Matters 7000. ■. Obtain sequencing read lengths of 400 to 500 bases.. ■. Generate more than 1 million sequencing reads per 10-hour instrument run.. ■. Improve performance by using GS FLX Titanium series reagents — without instrument upgrades.. ■. Accelerate the pace of discovery with easy-to-use analysis tools for straightforward interpretation of data and biologically meaningful results.. Modal read length. Mapped Reads. 6000 5000 4000 3000 2000 1000 0 0. 100. 200. 300. 400. 500. 600. Length (base pairs). Example Read Length Distribution of 629,643 reads from E. coli K-12 (Genome size ~4.5 Mb) with a modal read length of 504 bases.. Performance, Results, Impact Learn more at www.genome-sequencing.com. For life science research only. Not for use in diagnostic procedures. 454, 454 LIFE SCIENCES, 454 SEQUENCING, GENOME SEQUENCER, and GS FLX TITANIUM are trademarks of Roche. Other brands or product names are trademarks of their respective holders. © 2008 Roche Diagnostics GmbH. All rights reserved.. Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim, Germany.

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