Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
THE CAPPABLE-SEQ APPROACH FOR ANALYSIS OF
MICROBIAL TRANSCRIPTOMES
Wednesday, 1st June 18:30 La Fonda Mezzanine (2nd Floor) Poster (PS‐2.08)
Ira Schildkraut, Laurence Ettwiller, John Buswell, Erbay Yigit, Bo Yan
New England Biolabs, Inc.
The 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.
We 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, mapping reads to the bacterial genomes contained
in NCBI databases. We identified significant TSS signatures (>300 TSS) in many species and further
analyzed representative members of 4 different phyla. Reads mapping to rRNA and transfer
RNA (tRNA) represented less than 10 % of mappable reads indicating that Cappable‐seq depletes
processed transcripts such as rRNA and tRNA from microbiome total RNA. Cappable‐seq captures
the 5 end of primary transcripts enabling robust TSS determination in bacteria and microbiomes.
Furthermore, Cappable‐seq can reduce the complexity of the transcriptomes to quantifiable tags
enabling digital profiling of gene expression in any microbiome.
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