Abstract: Objective：We have upgraded the data analysis pipeline (Fudan pipeline 1.0) to Fudan pipeline 2.0 for high throughput sequencing which established in 2015. The purpose of this study is to compare the turn around time and accuracy of these two pipelines. Methods：In this study, 112 continuous samples from neonatal intensive care unit were recruited. Both Fudan pipeline 1.0 and 2.0 were used for the data analysis. We compared the results of preliminary analysis, time-consuming, and the accuracy between these two pipelines. And the main features of the Fudan pipeline 2.0 are explained in detail. Results：On the comparison of preliminary data analysis results, the variations to manual analysis step of Fudan pipeline 2.0 (an average of 25 variants) was significantly less than that of Fudan pipeline 1.0 (an average of 210 variants). On the comparison of efficiency, the turn around time of Fudan pipeline 2.0 (19.8h) was significantly shorter than that of Fudan pipeline 1.0 (78.8h). The diagnosis coincidence rate of Fudan pipeline 2.0 is 63.6% (7/11) for positive cases, and 84.2% (85/101) for negative cases with manual review. Conclusion：This study clearly shows the efficient, accurate and automated of Fudan pipeline 2.0 for genetic diagnosis with large sample size.