由於選擇性剪接機制的存在，基因轉錄後會形成多種的基因同種型(Gene Isoform)，而不同的基因同種型轉譯後形成不同蛋白質，可能具有不同的生理功能，我們認為針對基因同種型的表現分析比基因更為精確。基因同種型的分析可應用於計算兩狀態間(例如: 正常與癌症一期)的差異表現、表現高低影響病患存活時間的顯著性以及找出精確的癌症生物標記(Biomarker)等。利用RNA-Seq實驗技術搭配適當的分析流程可用於量化基因或基因同種型的表現，現今已普遍應用於癌症研究，我們資料庫採用Cufflinks軟體量化基因與基因同種型的表現，以及利用Cuffdiff計算不同狀態間的差異表現。目前有ISOexpresso資料庫與UALCAN資料庫提供使用者查尋基因或基因同種型的差異表現，但ISOexpresso只將資料區分成正常與癌症兩種類別，並沒有將癌症以期別細分，無法查詢基因或基因同種型在癌症不同期別之間的差異表現，UALCAN資料庫沒有對基因同種型層次分析差異表現，我們認為針對基因同種型層次的分析可以比基因層次更為精確且直接，綜合以上因素我們有了建立DEIso資料庫的想法。我們自TCGA收錄了519筆結腸癌與176筆直腸癌的比對後序列資料(Aligned BAM)，以及459筆結腸癌與166筆直腸癌的病人臨床資料，總資料量約4TB，將資料以癌症期別分類後，利用Cufflinks計算基因與基因同種型的表現量與差異表現倍率變化，並以簡單明瞭的視覺化圖視呈現搜尋結果，包括基因同種型結構圖、盒狀圖、熱區圖與存活分析圖。並以FOXQ1、ADAM8與SLC39A14三個我們資料庫篩選出具有差異表現的基因，表現趨勢皆與過去文獻實驗結果相同，因此相信我們資料庫的資料處理流程具有生物意義，此外，我們的資料庫預測CD44的三個外顯子差異可能可以做為癌症生物標記，值得生物學家進一步研究證實。DEIso資料庫網址: http://cosbi4.ee.ncku.edu.tw/DEIso/。

Alternative splicing events result in multiple gene isoforms, and it plays an important role in molecular mechanisms. Due to alternative splicing, we believe that the analysis of the isoform level is more accurate than the gene level. Gene isoforms analysis can be used to calculate the differential expression between the two conditions, the patient's survival analysis, and the identification of cancer biomarkers. RNA-Seq can be used to quantify the expression of genes or isoforms and it has been commonly used in cancer research. There are ISOexpresso and UALCAN databases that provide users with differential expression in a gene or isoform. However, ISOexpresso only classifies data as normal and cancer, UALCAN database does not analyze differential expression in isoform level. Based on the above reasons, we have the idea of establishing a database. We downloaded 519 BAM files of colon cancer and 176 BAM files of rectum cancer, and clinical data of 459 patients with colon cancer and 166 patients with rectum cancer. We classified all BAM files as normal, stage i, stage ii, stage iii, and stage iv, calculated expression and differential expression by Cufflinks, and presented search results with a visualization which is easy to understand, such as isoform structure, boxplot, heatmaps, and survival plot. We have the same trend after cross-validation with the results of the previous literatures. Therefore, we believe that the data processing flow of our database has significance, and the useful presentation will help cancer research.

[1] F. Crick,, "Central dogma of molecular biology.," Nature, vol. 227, no. 5258, pp. 561-563, 1970.
[2] A. Ramanathan, G. B. Robb, and S. H. Chan, "mRNA capping: biological functions and applications.," Nucleic Acids Research, vol. 16, pp. 7511-7526, 2016.
[3] D. Munroe, and A. Jacobson, "mRNA poly(A) tail, a 3' enhancer of translational initiation.," Molecular and Cellular Biology, vol. 7, pp. 3441-3455, 1990.
[4] N. J. Proudfoot, "Ending the message: poly(A) signals then and now.," Genes Development, vol. 25, no. 17, pp. 1770-1782, 2011.
[5] M. Chen, and J. L. Manley, "Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches.," Nature Reviews Molecular Cell Biology, vol. 10, no. 11, pp. 741-754, 2009.
[6] E. Park, Z. Pan, Z. Zhang, and L. L. YiXing, "The Expanding Landscape of Alternative Splicing Variation in Human Populations.," The American Journal of Human Genetics,, pp. 11-26, 2018.
[7] P. J Gardina, T. A Clark, B. Shimada, M. K Staples, Q. Yang, J. Veitch, A. Schweitzer, T. Awad, C. Sugnet, S. Dee, C. Davies, A. Williams, and Y. Turpaz, "Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array.," BMC Genomics, vol. 7, no. 325, 2006.
[8] M. Ganassi, S. Badodi, A. Polacchini, F. Baruffaldi, R. Battini, S. M. Hughesb, Y.Hinits, and S. Molinari, "Distinct functions of alternatively spliced isoforms encoded by zebrafish mef2ca and mef2cb.," Biochim et Biophysica Acta, vol. 1839, no. 7, pp. 559-570, 2014.
[9] T. C. Huang, P. T. Lee, M. H. Wu, C. C. Huang, C. Y. Ko, Y. C. Lee, D. Y. Lin, Y. W. Cheng, and K. H. Lee, "Distinct roles and differential expression levels of Wnt5a mRNA isoforms in colorectal cancer cells.," PLoSOne, vol. 12, no. 8, 2017.
[10] K. R. Kukurba, and S. B. Montgomery, "RNA Sequencing and Analysis.," Cold Spring Harbor Protocols,, vol. 2015, no. 11, pp. 951-969,, 2015.
[11] Z. Wang, M. Gerstein, and M. Snyder, "RNA-Seq: a revolutionary tool for transcriptomics.," Nature Review Genetics, vol. 10, no. 1, pp. 57-63, 2009.
[12] A. Yamada, P. Yu, W. Lin, Y. Okugawa, C. R. Boland, and A. Goel, "A RNA-Sequencing approach for the identification of novel long non-coding RNA biomarkers in colorectal cancer.," Scientific Report,, vol. 1, p. 575, 2018.
[13] I. S. Yang, H. Son, S. Kim, and S. Kim, "ISOexpresso: a web-based platform for isoform-level expression analysis in human cancer.,," BMC Genomics, vol. 17, no. 1, p. 631, 2016.
[14] D. S. Chandrashekar, B. Bashel, S. A. H. Balasubramanya, C. J. Creighton, I. P. Rodriguez, B. V. S. K. Chakravarthi, and S. Varambally,, "UALCAN: A Portal for Facilitating Tumor Subgroup Gene Expression and Survival Analyses.,," Neoplasia, vol. 19, no. 8, pp. 679-658, 2017.
[15] A. Dobin, C. A. Davis, F. Schlesinger, J. Drenkow, C. Zaleski, S. Jha, P. Batut, M. Chaisson, and T. R. Gingeras, "STAR: ultrafast universal RNA-Seq aligner.," Bioinformatics, vol. 29, no. 1, pp. 15-21, 2013.
[16] A. Dobin, and T. R. Gingeras, "Mapping RNA-Seq Reads with STAR.," Current Protocols2015, vol. 51, pp. 11.14.1-11.14.19, 2015.
[17] B. Li, and C. N. Dewey, "RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.," BMC Bioinformatics, p. 323, 2011.
[18] S. Anders, P. T. Pyl, and W. Huber, "HTSeq—a Python framework to work with high-throughput sequencing data.," Bioinformatics, vol. 2, pp. 166-167, 2015.
[19] M.D. Robinson, D.J. McCarthy, and G.K. Smyth, "edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.," Bioinformatics, vol. 26, no. 1, pp. 139-140, 2010.
[20] F. A. Haggar, and R. P. Boushey, "Colorectal Cancer Epidemiology: Incidence, Mortality, Survival, and Risk Factors.," Clinics in Colon and Rectal Surgery, vol. 4, pp. 191-197, 2009.
[21] Y. Yuan, M. D. Li, H. G. Hu, C. X. Dong, J.Q. Chen, X. F. Li, J. J. Li, and H. Shen, "Prognostic and survival analysis of 837 Chinese colorectal cancer patients.," World journal of Gastroenterology, vol. 19, no. 17, pp. 2650-2659, 2013.
[22] M. R. Hassan, M. A. Suan, S. A. Soelar, N. S.Mohammed, I. Ismail, and F Ahmad, "Survival Analysis and Prognostic Factors for Colorectal Cancer Patients in Malaysia.," Asian Pacific journal of cancer prevention, vol. 17, no. 7, pp. 3575-3581, 2016.
[23] C. Li, Z. Shen, Y. Zhou, and W. Yu, "Independent prognostic genes and mechanism investigation for colon cancer.," iological research, vol. 51, no. 1, p. 10, 2018.
[24] E. A. H. Kheirelseid, N. Miller, K. H. Chang, M. Nugent, and M. J. Kerin, "Clinical applications of gene expression in colorectal cancer.," Journal of Gastrointestinal Oncology, vol. 4, no. 2, pp. 144-157, 2013.
[25] M. J. Smith, A. C. Culhane, M. Donovan, J. C. Coffey, B. D. Barry, M. A. Kelly, D. G. Higgins, J. H. Wang, W. O. Kirwan, T. G. Cotter, and H. P. Redmond., "Analysis of differential gene expression in colorectal cancer and stroma using fluorescence-activated cell sorting purification.," Br J Cancer, vol. 100, no. 9, pp. 1452-1464, 2009.
[26] S. Anders, and W. Huber, "Differential expression analysis for sequence count data.," Genome Biology,, vol. 11, p. R106, 2010.
[27] P. Pitule, O. Vycital, J. Bruha, P. Novak, P. Hosek, V. Treska, I. Hlavata, P. Soucek, M. Kralickova, and V. Liska, "Differential expression and prognostic role of selected genes in colorectal cancer patients.," Anticancer Research, vol. 33, no. 11, pp. 4855-4865, 2013.
[28] H. Kaneda, T. Arao, K. Tanaka, D. Tamura, K. Aomatsu, K. Kudo, K. Sakai, D. Velasco MA, K. Matsumoto, Y. Fujita, Y. Yamada, J. Tsurutani, I. Okamoto, K. Nakagawa, and K. Nishio, "FOXQ1 is overexpressed in colorectal cancer and enhances tumorigenicity and tumor growth.,," Cancer Research, vol. 70, no. 5, pp. 2053-2063, 2010.
[29] Z. Yang, Y. Bai, L. Huo, H. Chen, J. Huang, J. Li, X. Fan, Z. Yang, L. Wang, and J. Wang, "Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer.," BMC cancer,, vol. 568, 2014.
[30] K. Thorsen, F. Mansilla, T. Schepeler, Bodil Øster, M. H. Rasmussen, L. Dyrskjøt, R. Karni, M. Akerman, A. R. Krainer, S. Laurberg, C. L. Andersen, and T. F. Ørntoft, "Alternative Splicing of SLC39A14 in Colorectal Cancer is Regulated by the Wnt Pathway.," Molecular & Cellular Proteomics, vol. 10, no. 1, p. M110.002998, 2010.
[31] A. Sveen, A. C. Bakken, T. H. Ågesen, G. E. Lind, A. Nesbakken, O. Nordgård, S. Brackmann, T. O. Rognum , R. A. Lothe, and R. I. Skotheim, "The exon‐level biomarker SLC39A14 has organ‐confined cancer‐specificity in colorectal cancer.," International Journal of Cancer, vol. 131, no. 6, pp. 1479-1485, 2011.
[32] L. Du, H. Wang, L. He, J. Zhang, B. Ni, X. Wang, H. Jin, N. Cahuzac, M. Mehrpour, Y. Lu, and Q. Chen., "CD44 is of functional importance for colorectal cancer stem cells.," Clinical Cancer Research, vol. 14, no. 23, pp. 7964-7967, 2008.
[33] A. C. Moss, G. Lawlor, D. Murray, D. Tighe, S. F. Madden, A. M. Mulligan, C. O. Keane, H. R. Brady, P. P. Doran, and P. MacMathuna., "ETV4 and Myeov knockdown impairs colon cancer cell line proliferation and invasion.," Biochemical and biophysical research communications, vol. 345, no. 1, pp. 216-221, 2006.
[34] S. Nakagawa, N. Miyoshi, H. Ishii, K. Mimori, F. Tanaka, M. Sekimoto, Y. Doki, and M. Mori., "Expression of CLDN1 in colorectal cancer: a novel marker for prognosis.,," International journal of oncology, vol. 39, no. 4, pp. 791-796, 2011.
[35] X. Wang, X. Liu, Angela Y. J. Li, L. Chen, L. Lai, H. H. Lin, S. Hu, L. Yao, J. Peng, S. Loera, L. Xue, B. Zhou, L. Zhou, S. Zheng, P. Chu, S. Zhang, D. K. Ann, and Y. Yen,, "Overexpression of HMGA2 Promotes Metastasis and Impacts Survival of Colorectal Cancers.," Clinical Cancer Research, vol. 8, pp. 2570-2580, 2011.
[36] C. Trapnell, A. Roberts, L. Goff, G. Pertea, D. Kim, D. R. Kelley, H. Pimentel, S. L. Salzberg, J. L. Rinn, and L. Pachter, "Distinct transcriptional regulatory modules underlie STAT3's cell type-independent and cell type-specific functions.," Nature Protocols, vol. 3, pp. 562-578, 2012.
[37] Y. Kang, M. Yin, W. Jiang, H. Zhang, B. Xia, Y. Xue, and Y. Huang, "Overexpression of LAPTM4B-35 is associated with poor prognosis in colorectal carcinoma.," ScienceDirect, vol. 204, no. 5, pp. 677-683, 2012.
[38] B. Alaiyan, N. Ilyayev, A. Stojadinovic, M. Izadjoo, M. Roistacher, V. Pavlov, V. Tzivin, D. Halle, H. Pan, B. Trink, A. O. Gure, and A. Nissan., "Differential expression of colon cancer associated transcript1 (CCAT1) along the colonic adenoma-carcinoma sequence.," BMC cancer, vol. 13, 2013.
[39] J. Christensen, S. Bentz, T. Sengstag, V. P. Shastri, and P. Anderle, "FOXQ1, a novel target of the Wnt pathway and a new marker for activation of Wnt signaling in solid tumors.," PLoS One, vol. 8, no. 3, p. e60051, 2013.
[40] H. MJ. Roelofs, R. HM. T. Morsche, B. WH. V. Heumen, F. M. Nagengast, and W. HM. Peters, "Over-expression of COX-2 mRNA in colorectal cancer.," BMC Gastroenterology, 2014.
[41] S. P. Fink, L. L. Myeroff, R. Kariv, P. Platzer, B. Xin, D. Mikkola, E. Lawrence, N. Morris, A. Nosrati, J. K. Willson, J. Willis, M. Veigl, J. S. Barnholtz-Sloan, Z. Wang, and S. D. Markowitz, "Induction of KIAA1199/CEMIP is associated with colon cancer phenotype and poor patient survival.," Oncotarget, vol. 6, no. 31, pp. 30500-30515, 2015.
[42] Y. Wang, J. Liu, B. Huang, Y. M. Xu, J. Li, L. F. Huang, J. Lin, J. Zhang, Q. H. Min, W. M. Yang, and X. Z. Wang, "Mechanism of alternative splicing and its regulation.," Biomedical Reports, vol. 3, no. 2, pp. 152-158, 2015.
[43] H. Jia, L. Song, Q. Cong, J. Wang, H. Xu, Y. Chu, Q. Li, Y. Zhang, X. Zou, C. Zhang, Y. E. Chin, X. Zhang, Z. Li, K. Zhu, B Wang, H. Peng, and Z. Hou, "The LIM protein AJUBA promotes colorectal cancer cell survival through suppression of JAK1/STAT1/IFIT2 network.," Nature, vol. 36, no. 19, pp. 2655-2666, 2016.
[44] G. Song, S. Xu, H. Zhang, Y. Wang, C. Xiao, T. Jiang, L. Wu, T. Zhang, X. Sun, L. Zhong, C. Zhou, Z. Wang, Z. Peng, J. Chen, and X. Wang, "TIMP1 is a prognostic marker for the progression and metastasis of colon cancer through FAK-PI3K/AKT and MAPK pathway.," Journal of Experimental & Clinical Cancer Research, vol. 35, no. 148, 2016.
[45] C. H. Lai, Y. C. Huang, J. C. Lee, J. T. C. Tseng, K. C. Chang, Y. J. Chen, N. J. Ding, P. H. Huang, W. C. Chang, B. W. Lin, R. Y. Chen, Y. C. Wang, Y. C. Lai, and L. Y. Hung, "Translational upregulation of Aurora-A by hnRNP Q1 contributes to cell proliferation and tumorigenesis in colorectal cancer.," Cell Death & Disease, vol. 8, no. 1, p. e2555, 2017.
[46] J. Peng, Q. Ou, J. Guo, Z. Pan, R. Zhang, X. Wu, Y. Zhao, Y. Deng, C. Li, F. Wang, L. Li, G. Chen, Z. Lu, P. Ding, D. Wan, and Y. Fang, "Expression of a novel CNPY2 isoform in colorectal cancer and its association with oncologic prognosis.," Aging,, vol. 9, no. 1, pp. 2334-2351, 2017.
[47] J. Sun, X. Liu, H. Gao, L. Zhang, Q. Ji, Z. Wang, L. Zhou, Y. Wang, H. Sui, Z. Fan, and Q Li, "Overexpression of colorectal cancer oncogene CHRDL2 predicts a poor prognosis.,," Oncotarget, vol. 8, no. 7, pp. 11489-11506, 2017.
[48] V. Vaish, J. Kim, and M. Shim, "Jagged-2 (JAG2) enhances tumorigenicity and chemoresistance of colorectal cancer cells.," Oncotarget, vol. 8, no. 32, pp. 53262-53275, 2017.
[49] C. K. Tsai, L. C. Huang, W. C. Tsai, S. M. Huang, J. T. Lee, and D. Y. Hueng, "Overexpression of PLOD3 promotes tumor progression and poor prognosis in gliomas.," Oncotarget, vol. 9, no. 21, pp. 15705-15720,, 2018.
[50] C. Li, D. R. Liu, L. Y. Ye, L. N. Huang, S. Jaiswal, X. W. Li, H. H. Wang, and L. Chen, "HER-2 overexpression and survival in colorectal cancer: a meta-analysis.," Journal if Zhejiang University SCIENCE B, vol. 15, no. 6, pp. 582-589, 2018.
[51] A. Mezheyeuski, C. Strell, I. Hrynchyk, T. K. Guren, A. Dragomir, T. Doroshenko, O. Pashkova, J. Gorgun, K. Ruksha, P. Pfeiffer, H. Kure E, H. Sorbye, D. Edler, A. Martling, B. Glimelius, A. Östman, and A. Portyanko, "Treatment-related survival associations of claudin-2 expression in fibroblasts of colorectal cancer.," Virchows Archiv, vol. 472, no. 3, pp. 395-405, 2018.
[52] J. Zhang, Y. Jiang, J. Zhu, T. Wu, J. Ma, C. Du, S. Chen, T. Li, J. Han, and X. Wang, "Overexpression of long non-coding RNA colon cancer-associated transcript 2 is associated with advanced tumor progression and poor prognosis in patients with colorectal cancer.," ONCOLOGY LETTERS, vol. 14, no. 6, pp. 6907-6914, 2017.