Computational protocol: RNA-seq reveals the critical role of OtpR in regulating Brucella melitensis metabolism and virulence under acidic stress

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Protocol publication

[…] To increase the quality of the reads, the raw reads with the length of 90-bp each were trimmed to 75 bp after quality evaluation using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The trimmed reads were aligned with the B. melitensis 16 M genome (NC_003317 and NC_003318) and annotated gene sets obtained from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/) using the Short Oligonucleotide Analysis Package (SOAP). cDNAs with matches to the reference genome of >80% were retained for further analysis. Those sequencing reads that matched annotated genes in the B. melitensis 16 M genome reflect the genes transcribed under the given experimental conditions. Gene expression was quantified as Reads Per Kilobase of coding sequence per Million reads RPKM algorithm. A gene was considered to be differentially expressed if the difference in RPKM values between the two samples (16 M and 16 MΔotpR) was ≥2.0-fold (i.e., log2 ratio >1.0) and the p-value was <0.05. […]

Pipeline specifications

Software tools FastQC, SOAP
Application RNA-seq analysis
Organisms Brucella melitensis, Brucella melitensis bv. 1 str. 16M
Chemicals Carbon, Iron, Nitrogen