Construction Of Different RNA-seq Analysis Platforms And Their Application In Somatic Cell Reprogramming Gene Differential Expression Analysis

RNA-seq is a common method to study gene expression.At present,there are nearly 100 kinds of softwares to analyze RNA-seq data.This study is based on the same hardware equipment,using the traditional Tophat2 software combination and the new development of HISAT2 software combination to build two sets of RNA-seq data analysis platforms and make a comparison.The results show that the computing time of HISAT2 is about 5 times faster than that of Tophat2,the maximum memory is 1GB samller than that of Tophat2,and the alignment rate is 5%to 6%higher than that of Tophat2.HISAT2 can aligne more reads to the genome.The computing time of Stringtie is about 2 to 3 times faster than that of Cufflinks,the maximum memory of Stringtie is about 1/6 to 1/7 of Cufflinks during the process,and Stringtie assembles more 854 transcripts and 109 genes than Cufflinks.To analysis the same data,Ballgown requires 3s,while Cuffdiff2 needs nearly 2 days,the maximum memory Ballgown occupies only 1/78 Cuffdiff2,The number of differential expressed genes Ballgown detecting is about two times of Cuffdiff2 detecting.Overall,the total time of HISAT2 software platform is 10h3min4s and maximum memory is 4.279GB,while the total time of Tophat2 software platform is about 4 days and the maximum memory is 163.84GB.The new analysis platform can not only save a lot of time,but also improve the sensitivity of analysis.Further,we compared the 3 pluripotent stem cells,NT,iPS,hES cells,and made differential expressed genes analysis,GO analysis,KEGG analysis and genes'interaction network analysis.Results show that the number of up-regulated differential expressed genes between iPS cells and hES cells is 961,most of which are significantly enriched to the transcriptional regulation of DNA in the process,while the number of down-regulated genes is 577,most of which are significantly enriched to endocytosis pathway;The number of up-regulated differential expressed genes between NT cells and hES cells is 124,down-regulated differential expressed genes number is 104,most which is significantly enriched in innate immune response.The number of up-regulated differential expressed genes between NT cells and iPS cells was 1153,and most of the genes are significantly enriched in pathway about regulating pluripotent stem cells,suggesting that NT cells are superior to iPS cells in the aspect of pluripotent.The number of down-regulated differential expressed genes between NT cells and iPS cells was 647.Summarize the results of differential expressed genes analysis and enrichment analysis,NT cells and hES cells were more similar,comparing with iPS cells and hES cells.Then we compared NT,iPS,hES and HDF cells,and found some pathways related to reprogramming,like DNA synthesis and repair,stem cell pluripotent maintenance,regulation of mRNA stability and so on.This study provides some theoretical basis for further revealing the mechanism of reprogramming.