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

BMC Genomics

Methodology Article

Erbay Yigit¹  Ira Schildkraut¹  Laurence Ettwiller¹  John Buswell¹ 

[1] New England Biolabs, 240 County Rd, 01938, Ipswich, MA, USA;

关键词: Transcriptome;    Transcription start sites;    Microbiome;   

DOI  :  10.1186/s12864-016-2539-z

 received in 2015-11-03, accepted in 2016-02-25,  发布年份 2016

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© Ettwiller et al. 2016