Sperm MRNA High-throughput Sequencing And Detection Result Evaluation

Traditional theory of embryonic development holds that after meiosis, round spermatid develops into mature sperm through metamorphosis, the spermatid chromatin is stringently packaged and highly concentrated, and most cytoplasm in the germ cell is gradually lost. So it was thought that the transcription activity of spermatid is lost and RNAs are absent in spermatid, and just deliver genome to the eggs. So in early embryonic development process just maternal factor play a role. However with the development of technology and the deepening of the study, sperm RNA were detected in recent years. But there is no whole pig sperm gene expression profiles, and few studies on the origin of sperm RNA now. In this experiment, the ejaculated pig sperm was used for the study. PureSperm centrifugation was applied to separate and collect high quality sperms. High-throughput sequencing was processed using this sample, then we get the sequence data. Quality control was done with FastQC first. Then the sequence data were mapped to the genome with Tophat. With Tophat alignment results, assembly and expression quantification were processed by using cufflinks. Highly expressed genes are then selected using the DAVID software for functional annotation analysis. Finally, RT-PCR experiments were done to verify the expression of several genes. Through the above research established a method for collecting a sample of sperm, sperm mRNA high-throughput sequencing and data analysis. To further study the origin and screening of key genes of sperm RNA research laid the foundation.Results:1. The method to collect sperm samples is effective, and were confirmed to have a high purity.2. RNA were exacted from ejaculated sperm samples, and mRNA sequence database was built. We got 54860218 total reads and 54001227 clean reads after high-throughput sequence.3. Clean reads were mapped to the genome, and got the 63% mapping ratio. Then assembly the mapping result and quantitative the expression. Then we got 12073 genes with FPKM≥1, and 1025 genes with FPKM^50 and official gene symbol. Then these 1025 genes were used for functional analysis, and 249 genes were well annotated. By use DAVID we got 22 GO terms with more than 10 genes in each term. And 8 pathways in KEGG database.4. By RT-PCR on AQN-1, AQN-3, AWN, PSP-I, PSP-II gene expression validation test results consistent with the sequencing results.By using sperm floating and gradient centrifugation method, we have successfully established a complete set of valid sperm sample collection and purification scheme. Sperm mRNA was successfully performed high-throughput sequencing, enabling us to grasp the sperm RNA high-throughput sequencing technology. Through quality testing and finished of the sequence data analysis, made us familiar with and master the initial mRNA sequencing data analysis process. Laid the foundation for further in-depth study of the origin and function of sperm RNA.