iRNA-seq: computational method for genome-wide assessment of acute transcriptional regulation from total RNA-seq data.
Bottom Line: RNA-seq is a sensitive and accurate technique to compare steady-state levels of RNA between different cellular states.However, as it does not provide an account of transcriptional activity per se, other technologies are needed to more precisely determine acute transcriptional responses.In conclusion, iRNA-seq offers an attractive novel alternative to current methods for determination of changes in transcriptional activity at a genome-wide level.
Affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M 5230, Denmark NNF Center of Basic Metabolic Research, University of Copenhagen, Copenhagen 2200, Denmark.Show MeSH
Mentions: To investigate this at a genome-wide level, we designed a Perl pipeline, iRNA-seq, which for every gene quantifies and sums intron reads not overlapping with any other transcripts or non-coding RNA. Intron reads are counted and summed using featureCounts (23), and differential expression analysis is performed using edgeR (24) (Figure 2A). To minimize problems arising from inclusion of exon reads due to differential exon usage (Figure 2B, arrow A) and incomplete annotation (Figure 2B, arrow B), all regions associated with mRNA were subtracted from the regions subjected to counting (see Materials and Methods). In addition to the default quantification of intron reads, the iRNA-seq tool also allows quantification of reads in exons or reads in full-length genes. Thus, the iRNA-seq tool allows fast and easy parallel assessment of mature transcript levels as well as active transcription from one total RNA-seq experiment.
Affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M 5230, Denmark NNF Center of Basic Metabolic Research, University of Copenhagen, Copenhagen 2200, Denmark.