Computational protocol: “Tuberculosis in advanced HIV infection is associated with increased expression of IFNγ and its downstream targets”

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Protocol publication

[…] RNA was isolated from whole blood using the PAXgene Blood RNA Kit (Qiagen, catalog # 762164). Total RNA input was amplified using MessageAmp™ II aRNA Amplification Kit (ThermoFisher Scientific). 100 ng of amplified RNA was used to prepare the library. Following standard instructions for fragmentation, purification, 3′ and 5′ adapter ligation and reverse transcription, PCR amplicons were purified using AMPure XP beads and the library was quantified. This library was used for RNA Sequencing on Illumina Hiseq2500. On average 49–50 million basepair (bp) reads were produced per sample. Quality check of reads was performed with the software FastQC. Low quality reads and adapters were trimmed with Cutadapt []. Trimmed reads were mapped to the human genome GRCh38/hg19 with STAR [], and the expression level for each gene was counted with HTSeq [] according to gene annotations from Ensembl. The Bioconductor DESeq package in R [] was used to normalize the counts and call differential expressions. Principal Component Analysis (PCA) was used for data visualization. Hierarchical clustering was performed with the gplots package in R. The ROC curve was plotted with the pROC package in R. Functional and network analyses of differentially expressed genes were performed using Ingenuity Pathway Analysis (IPA, Ingenuity Systems, https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/, Redwood City, CA). […]

Pipeline specifications

Software tools FastQC, cutadapt, STAR, HTSeq, DESeq, gplots, IPA
Application RNA-seq analysis
Diseases Infection, Lung Diseases, Tuberculosis, HIV Infections