A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome

Abstract

BackgroundThe initiating nucleotide found at the 5’ end of primary transcripts has a distinctive triphosphorylated end that distinguishes these transcripts from all other RNA species. Recognizing this distinction is key to deconvoluting the primary transcriptome from the plethora of processed transcripts that confound analysis of the transcriptome. The currently available methods do not use targeted enrichment for the 5′end of primary transcripts, but rather attempt to deplete non-targeted RNA.ResultsWe developed a method, Cappable-seq, for directly enriching for the 5' end of primary transcripts and enabling determination of transcription start sites at single base resolution. This is achieved by enzymatically modifying the 5′ triphosphorylated end of RNA with a selectable tag. We first applied Cappable-seq to E. coli, achieving up to 50 fold enrichment of primary transcripts and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. We also applied Cappable-seq to a mouse cecum sample and identified TSS in a microbiome.ConclusionsCappable-seq allows for the first time the capture of the 5′ end of primary transcripts. This enables a unique robust TSS determination in bacteria and microbiomes. In addition to and beyond TSS determination, Cappable-seq depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per transcript enabling digital profiling of gene expression in any microbiome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2539-z) contains supplementary material, which is available to authorized users.

Global repositioning of transcription start sites in a plant-fermenting bacterium

“…The 5′-PPP of mRNA are modified by vaccinia capping enzyme (VCE) to bear a biotinylated guanosine cap that facilitates their capture and purification using streptavidin magnetic beads. Recently, TSS were identified by Cappable-Seq14 using VCE to add a desthiobiotin cap for bead-based capture of 5′ mRNA, which were then eluted from the beads and de-capped to ligate adapters for reverse transcription to tagged cDNA. Capp-Switch streamlines this approach by reverse transcribing the 5′ mRNA fragments using template-switching by Moloney murine leukemia virus reverse (MMLV) transcriptase15.…”

Deciphering the Rules Underlying Xenogeneic Silencing and Counter-Silencing of Lsr2-like Proteins Using CgpS of Corynebacterium glutamicum as a Model

“…Determination of TSS. The determination of TSS and data analysis were performed with C. glutamicum wild-type cells by Vertis Biotechnology AG (Freising, Germany) using the Cappable-seq method developed by Ettwiller and Schildkraut (68). Prophage induction was triggered by adding mitomycin C. Detailed information about cultivation conditions, RNA preparation, and data analysis can be found in the supplemental material (Text S1).…”

Riboregulation in Nitrogen-Fixing Endosymbiotic Bacteria

“…Inherent limitations to both computational searches and microarray designs necessarily biased these early genome-wide screens for sRNAs in rhizobia to the identification of putative intergenic, trans-acting, conserved riboregulators. Straightforward experimental identification of TSS associated to coding sequences, untranslated mRNA regions, and non-coding RNA genes in the prokaryotic genomes is now feasible with the implementation of oriented differential RNA-Seq (dRNA-Seq) [24] or Cappable-Seq [25] strategies. In particular, dRNA-Seq surveys rediscovered the early-identified sRNAs in S. meliloti and B. japonicum and further uncovered the complex rhizobial transcriptomes by adding hundreds of unknown trans-sRNAs, as well as thousands of mRNA-derived sRNAs and asRNAs [22,[26][27][28][29].…”