Computational protocol: Comparative transcriptome analysis of peripheral blood mononuclear cells in hepatitis B-related acute-on-chronic liver failure

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Protocol publication

[…] The sequencing reads were mapped against hg38 using STAR (v2.4.0), which is an ultrafast universal RNA-seq aligner. All parameters were set to the default values except for the allowed maximum mismatch, which was set to 5% for each read, and the output of the BAM files, which were sorted by coordinate. The transcripts were assembled de novo using Cufflinks (v2.2.1), and the novel built GTF file was merged with the GTF file of hg38. With the merged GTF file, the FPKM of each transcript was estimated using the cuffquant and cuffnorm tools contained in the Cufflinks suite. The differential expression of each transcript and each gene was analysed using cuffdiff, which is also a component of Cufflinks. Condition-specific genes were also identified with cummeRbund by estimating the Jensen-Shannon distance. The significantly upregulated or downregulated genes in HBV-ACLF were then submitted to The Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.7) for GO enrichment analyses. The enriched GO terms with a false discovery rate (FDR) of <0.05 were considered significant and further subjected to a cluster analysis. [...] All statistical analyses were performed with the R software package. The significant DEGs and transcripts were analysed using the Cuffdiff and cummeRbund package. Unsupervised hierarchical clustering of the samples from the different groups with significant DEGs was performed using the ‘heatmap.2’ function contained in the gplots package. The DEGs validated via qRT-PCR were identified using Student’s t-test. ROC analysis was performed using the pROC package. A P-value of <0.05 was considered to indicate a significant difference. […]

Pipeline specifications

Software tools STAR, Cufflinks, CummeRbund, DAVID, gplots
Applications RNA-seq analysis, Transcriptome data visualization
Organisms Homo sapiens, Hepatitis B virus
Diseases Liver Failure, Acute, Hepatitis B, Chronic, Acute-On-Chronic Liver Failure