RNA-Seq-Based Comparative Transcriptomics: RNA Preparation and Bioinformatics
The major transcriptome analysis is the determination of differentially expressed genes across experimental conditions. For this, the next-generation sequencing of RNA (RNA-seq) is an increasingly cost-effective technology for the analysis of transcriptomes with several advantages over gene expression microarrays, such as its higher sensitivity and accuracy, broader dynamic range, and the ability to detect novel transcripts, including noncoding RNA molecules, at nucleotide-level resolution. Although these advantages, many microbiology laboratories have not yet applied RNA-seq analyses to their investigations. The high cost of the equipment for next-generation sequencing is no longer an issue, since this intermediate part of the analysis can be provided by commercial or central services. Here, we detail a protocol for the first part of the analysis, the RNA extraction, and an introductory protocol to the bioinformatics analysis of the sequencing data that generates the differential expression results.
Key wordsMycobacterium B-3805 Next-generation sequencing RNA extraction Transcriptomics RNA-seq Bioconductor Differential expression
This work was supported by a grant of the European Union program ERA-IB [MySterI (EIB.12.010)] through the APCIN call of the Spanish Ministry of Economy and Competitiveness (MINECO, Spain) (PCIN-2013-024-C02-01). The authors want to thank the European Union program ERA-IB; the Spanish Ministry of Economy and Competitiveness (MINECO, Spain) and the MySterI Consortium (INBIOTEC, Pharmins Ltd., University of York, SINTEF, Technische Universität Dortmund and Gadea Biopharma S.L.). We thank J. Merino, B. Martín, and A. Casenave for their excellent technical assistance and the degree students of the group A. Morales and P. González.