Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

Georgia Giannoukos,Andreas Gnirke,Chad Nusbaum,Joshua Z Levin,Doyle V Ward,Ashlee M Earl,Bruce W Birren,Dawn M Ciulla,Jacques Izard,Dirk Gevers,Jonathan Livny,Brian J Haas,Katherine Huang

doi:10.1186/gb-2012-13-3-r23

Georgia Giannoukos, Andreas Gnirke + Show 11 more

Open Access

https://doi.org/10.1186/gb-2012-13-3-r23

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Journal: Genome Biology	Publication Date: Jan 1, 2012
Citations: 247	License type: cc-by

Affiliation: Broad Institute, Harvard University

Abstract

We have developed a process for transcriptome analysis of bacterial communities that accommodates both intact and fragmented starting RNA and combines efficient rRNA removal with strand-specific RNA-seq. We applied this approach to an RNA mixture derived from three diverse cultured bacterial species and to RNA isolated from clinical stool samples. The resulting expression profiles were highly reproducible, enriched up to 40-fold for non-rRNA transcripts, and correlated well with profiles representing undepleted total RNA.

Highlights

Microbial communities are known to play significant roles in human health, development, and disease [1,2,3,4], and DNA sequencing is an effective approach to characterize the structure and potential function of these communities
Evaluation of ribosomal RNA (rRNA) depletion methods To provide a benchmark for method evaluation, we prepared RNA from three well characterized organisms (Prochlorococcus marinus, Escherichia coli, and Rhodobacter sphaeroides) that cover a wide range of base compositions (30%, 51%, and 69% genome GC content, respectively)
We constructed RNA-seq libraries from total RNA and rRNA-depleted samples using each of the methods, sequenced them using the Illumina platform, mapped the reads to the three reference genome sequences and separately counted reads that aligned to rRNA and to the coding DNA sequence (CDS) of annotated genes

Summary

Introduction

Microbial communities are known to play significant roles in human health, development, and disease [1,2,3,4], and DNA sequencing is an effective approach to characterize the structure and potential function of these communities. Ultra-high throughput sequencing of transcriptomes, RNA-seq, has rapidly become the method of choice for revealing functional genes and pathways in individual microbes [8,9,10,11,12,13,14,15,16], as well as in complex environmental communities - for example, from the sea [17,18] and from the human gut [19,20]. Subtractive hybridization with non-commercial, samplespecific anti-rRNA probes increased the percentage of non-rRNA reads from phytoplankton RNA no more than about four-fold to slightly less than 50% [22]

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