Computational protocol: Rhinovirus infection results in stronger and more persistent genomic dysregulation: Evidence for altered innate immune response in asthmatics at baseline, early in infection, and during convalescence

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Protocol publication

[…] RNA was isolated from the nasal epithelial samples at the University of Virginia and sent to the University of Cincinnati College of Medicine Genomics, Epigenomics, and Sequencing Core for analysis via RNA-seq. Sequence reads were aligned to the reference human genome (hg19) using the TopHat aligner []. Reads aligning to each known transcript were counted and all follow up analyses were performed using Bioconductor packages for next-generation sequencing data analysis []. The differential gene expression analysis was performed based on the negative-binomial statistical model of read counts as implemented in the edgeR Bioconductor package []. Genes differentially expressed between asthma and control samples before inoculation (time point T0) were identified using the simple generalized linear model with the single factor (asthma = yes or no). Genes differentially expressed after inoculation (time points T1 and T2) in comparison to the baseline (time point T0) were identified by fitting a generalized linear model with both time and subject factors to account for the subject-to-subject variability. These comparisons were made separately for asthma and control samples. P-values were adjusted for multiple testing using the false discovery rates and differential expressions with FDR-adjusted p-value of less than 0.05 were considered statistically significant []. To elucidate the most common pathways affected the by gene dysregulation, all genes dysregulated at least 1.5 fold were analyzed with QIAGEN’s Ingenuity® Pathway Analysis (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity). […]

Pipeline specifications

Software tools TopHat, edgeR, IPA
Application RNA-seq analysis
Organisms Human poliovirus 1 Mahoney, Homo sapiens