Computational protocol: RNA-Seq-Based Analysis of the Physiologic Cold Shock-Induced Changes in Moraxella catarrhalis Gene Expression

Similar protocols

Protocol publication

[…] The quality analysis of RNA-seq data was carried out using the FastQC application (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The M. catarrhalis O35E genome sequence as well as open reading frame (ORF) positions and annotations were obtained using the RefSeq and PATRIC database (http://patricbrc.vbi.vt.edu/portal/portal/patric/Home and http://brcdownloads.vbi.vt.edu/patric2/genomes/Moraxella_catarrhalis_O35E). Reads were mapped to the M. catarrhalis O35E RefSeq genome sequence using the BWA algorithm . The summarization of counts was performed using the BEDTools software , and normalization and the differential expression analysis was performed using the variance analysis package DEseq using R version 2.8.0. The total read count was determined for each gene by combining data from three biological replicate sequencing runs. P-values were calculated and adjusted for multiple testing using the false discovery rate (FDR) controlling procedure . Functional classification of the M. catarrhalis O35E genes was projected from the M. catarrhalis RH4 genome by identifying the orthologous proteins between these strains using the best reciprocal blast hit approach with protein sequences. For all but seven proteins from the O35E strain an 1∶1 orthologous protein could be identified in the RH4 strain. The seven O35E proteins were equally similar to multiple (2 or 3) different RH4 proteins. Enrichment analysis was performed using the functional categories with a custom python skript (Fisher’s exact test) correcting for multiple testing using the FDR.http://www.nature.com/nature/journal/v486/n7402/full/nature11234.html - ref12. […]

Pipeline specifications

Software tools FastQC, BWA, BEDTools, DESeq
Databases PATRIC
Application RNA-seq analysis
Organisms Moraxella catarrhalis, Homo sapiens
Diseases Respiratory Tract Infections
Chemicals Iron, Nitrogen