iRNA-seq: computational method for genome-wide assessment of acute transcriptional regulation from total RNA-seq data.
Bottom Line: However, as it does not provide an account of transcriptional activity per se, other technologies are needed to more precisely determine acute transcriptional responses.Here, we have developed an easy, sensitive and accurate novel computational method, IRNA-SEQ: , for genome-wide assessment of transcriptional activity based on analysis of intron coverage from total RNA-seq data.However, unlike the current methods that are all very labor-intensive and demanding in terms of sample material and technologies, iRNA-seq is cheap and easy and requires very little sample material.
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.