Data Analysis And Quality Control Of High-throughput Single Cell Sequencing

High throughput single-cell RNA sequencing is a technique to detect gene expression at the single-cell level for studying cell heterogeneity,including automated single-cell sequencing and high-throughput data analysis.The former first realized high-throughput single-cell separation and automated single-cell sequencing library preparation based on "water-in-oil" emulsion technology and microfluidic technology;then realized high-throughput DNA sequencing based on the next-generation sequencing technology with modular mechanical firmware and highly automated operation process.The latter refers to that the massive data,which generated from the improved information density and capacity of experimental data due to the application of automation technology,is rely on biological knowledge and computer information technology to find the complex and diverse transcriptome landscape differences within the cell population.In this study,we used high-throughput single-cell RNA sequencing technology to study the long-term health hazards of low-dose bisphenol A(BPA)exposure based on human mesenchymal stem cell(MSC)model.Human MSCs were exposed to BPA to establish a long-term cytotoxicity model of low-dose BPA.The transcriptome data of the cytotoxicity model were obtained by high-throughput single-cell RNA sequencing.In this paper,we established a 10 x single cell data analysis workflow with Cell Ranger,Loupe Browser and a variety of analysis tools,and then used the workflow to complete the comparison,quantification and quality control of sequencing data.Firstly,the proportion of Q30 base in BPA group and control group was 85.3% and 86.4% respectively,which reached the 75% quality control standard of Illumina.Then,as the mapped confidently to transcriptome is a key index to evaluate the mapping quality,the transcript confidently mapping rates of BPA group and control group were 71.4% and 74.0%,respectively,which were much higher than 30% of the minimum standard requirements.Finally,the quality control analysis of cell characteristics was carried out,and the correlation analysis of various cell characteristics showed that more than 98% of the cell mitochondria accounted for less than 10%.In conclusion,the gene expression matrix obtained from upstream analysis can be used for downstream data analysis through quality control.Then the clustering results were analyzed by a variety of bioinformatics analysis tools,and the integration,annotation and functional analysis of cell subsets were completed according to the bioinformatics of each cell subsets.Firstly,the dimension reduction and clustering of the data are processed to get 16 Clusters.Then go analysis was used to characterize the function of cell population and quasi time analysis was used to reconstruct the development process of mesenchymal stem cells.According to the similarity of biological functions,16 Clusters were integrated into 8 Clusters,and the new Cluster differential genes were recalculated.Go analysis was used to comprehensively characterize the function of new Clusters,and the annotation of Clusters in development and differentiation was completed according to the results of go analysis.Then the GO terms related to immunity in the go analysis results of each Cluster were counted,and it was found that Cluster 1 and Cluster 8 were two cell subpopulations mutually exclusive in immune response.Through a detailed analysis of the GO terms of Cluster 1 and Cluster 8,we found that Cluster 1 is a subset of cells related to the activation of immune response,especially inflammation,and Cluster 8 is in the resting state of immunomodulator.After the analysis of development differentiation and immune regulation ability,the proportion of each Cluster in the two sample groups was counted.By comparing the proportion of functional cell subsets in the two sample groups,it was revealed that long-term exposure to low-dose BPA led to impaired stemness,enhanced adipogenic differentiation and immunomodulation disorder of MSCs.In conclusion,based on automatic control technology and large-scale data analysis,this paper established a workflow for data analysis of single-cell RNA sequencing data of mesenchymal stem cells exposed to BPA,and mined the biological information of the effect of long-term low-dose BPA exposure on mesenchymal stem cells.The analysis ideas of this paper is significant as the reference for the application of automation technology in the field of life science and environmental health.The results of this paper can be used as the risk assessment data of BPA environmental exposure to provide theoretical support and practical guidelines for the formulation of relevant policies and pollution control.