使用RNA-Seq資料分析策略解析尼羅吳郭魚（Oreochromis niloticus）之性別決定遺傳因子

Abstract

本研究以台灣重要具高經濟價值的養殖魚種吳郭魚（Oreochromis niloticus）為對象，以高效準確的生物資訊分析工具組成單點變異分析和表現量差異分析平台，尋找與吳郭魚性別決定有關的遺傳因子，作為後續吳郭魚性別分化研究的基礎。本研究分析發育第四天與第五天的吳郭魚苗雌雄胚胎的RNASeq。原始讀序經過清理、定址於吳郭魚基因體組裝序列（ASM185804v2）。選定雙端皆能被定址的讀序進行變異點分析，得到八組樣本共1,184,624個變異位點。觀察這些變異位點的組別與密度分布，並進行交叉比對以及關聯分析，篩選出獨屬雄性、雌性，以及在雌雄之間有差異的變異位點。我們觀察到LG 2在2.14~2.2Mbp區域的性別差異變異位點有明顯的集中現象，註解後得知所屬基因以及其造成的影響，配合使用GO分析、KEGG分析，發現這些基因可能參與了訊息傳導、細胞間交互作用、有絲分裂等反應。由雌雄基因表現量分析，得到5,129個性別差異表現基因。將性別相關變異位點分析中所得到2,136個基因與性別差異表現的基因名單交叉比對，找到277個基因在雌雄之間使用不同剪切位的轉錄序列，這些基因或許在性別分化歷程中扮有角色。本研究期望能對吳郭魚性別分化系統帶來更深入的了解，也希望在此研究中建立的高效分析平台，能協助其他物種進行類似的序列分析工作。

Nile Tilapia (Oreochromis niloticus) is one of the most economically valuable aquaculture species in Taiwan, but the mechanism of sex differentiation hasn’t been fully studied. In this study, we combine gene expression and single nucleotide variation analysis as main strategy to find variants that may be associated to tilapia’s sex differentiation process. A two replicates RNA-Seq experiment were done in four experimental conditions, male and female tilapia embryos in day 4 and day 5 respectively. Raw reads of 8 samples was cleaned and mapped with ASM185804v2 assembly. 1,184,624 single base variation sites were detected in 8 samples. We found a significant sex-biased SNV density increase in 2.14~2.20Mbp region of Linkage Group 2. Functional enrichment analysis of genes reside this sex-biased SNV region highlighted some GO categories and KEGG pathways, such as signal transduction, cell adherent, mitotic process, and etc. Gene expression analysis between 4 sets of samples revealed 5,129 differentially expressed genes between male and female. Comparing with the result of 2,136 genes with sex-biased SNV, 277 genes were identified to be translated in gender specific transcript preference. Some of these genes are also supported by the high expression level in gonads tissue like testis or ovary. These genes may probably be the candidate genes which involved in sex differentiation process. The NGS tools with high efficiency and accuracy were allocated to form a high-performance data analysis docker platform. We hope to contribute to other species’s NGS analysis studies through this established high-efficiency docker analysis platform.