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

Genome Sequencing and Analysis Program, The Broad Institute of MIT and Harvard, Cambridge, MA 02141, USA.
Genome biology (Impact Factor: 10.81). 03/2012; 13(3):R23. DOI: 10.1186/gb-2012-13-3-r23
Source: PubMed


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.

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    • "Giannoukos et al. presented a protocol for metatranscriptomic analysis of bacterial communities that accommodates both intact and fragmented RNA and combines efficient rRNA removal with strand-specific RNA-seq (Giannoukos et al., 2012). Currently, only a handful of metatranscriptomic datasets are available (and metaproteomic datasets are even scarcer), but we envision a flood of metatranscriptomic data in the near future, as experimental techniques mature (Giannoukos et al., 2012; Franzosa et al., 2014). Metatranscriptome analyses typically include the assignment of the predicted function and taxonomic origin of RNA-seq reads, by directly searching metatranscriptomic sequences (bags of reads) against prokaryotic genomes (the reference genomes) (Leimena et al., 2013) or known protein sequences (Franzosa et al., 2014). "
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    ABSTRACT: Metagenomics research has accelerated the studies of microbial organisms, providing insights into the composition and potential functionality of various microbial communities. Metatranscriptomics (studies of the transcripts from a mixture of microbial species) and other meta-omics approaches hold even greater promise for providing additional insights into functional and regulatory characteristics of the microbial communities. Current metatranscriptomics projects are often carried out without matched metagenomic datasets (of the same microbial communities). For the projects that produce both metatranscriptomic and metagenomic datasets, their analyses are often not integrated. Metagenome assemblies are far from perfect, partially explaining why metagenome assemblies are not used for the analysis of metatranscriptomic datasets. Here we report a reads mapping algorithm for mapping of short reads onto a de Bruijn graph of assemblies. A hash table of junction k-mers (k-mers spanning branching structures in the de Bruijn graph) is used to facilitate fast mapping of reads to the graph. We developed an application of this mapping algorithm: a reference based approach to metatranscriptome assembly using graphs of metagenome assembly as the reference. Our results show that this new approach (called TAG) helps to assemble substantially more transcripts that otherwise would have been missed or truncated because of the fragmented nature of the reference metagenome. TAG was implemented in C++ and has been tested extensively on the linux platform. It is available for download as open source at http://omics.informatics.indiana.edu/TAG. yye@indiana.edu. © The Author(s) 2015. Published by Oxford University Press.
    Bioinformatics 04/2015; DOI:10.1093/bioinformatics/btv510 · 4.98 Impact Factor
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    • "D: Alphaproteobacterial RNA example originating from Rhodobacter spaeroides (NC_011963.1) showing putative transcription start site determined from analysis of RNA-seq reads (Additional file 4: Figure S1A) [34]. E: Actinobacterial RNA example originating from Mycobacterium tuberculosis (NC_000962.3) "
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    ABSTRACT: Autogenous cis-regulators of ribosomal protein synthesis play a critical role in maintaining the stoichiometry of ribosome components. Structured portions within an mRNA transcript typically interact with specific ribosomal proteins to prevent expression of the entire operon, thus balancing levels of ribosomal proteins across transcriptional units. Three distinct RNA structures from different bacterial phyla have demonstrated interactions with S15 to regulate gene expression; however, these RNAs are distributed across a small fraction of bacterial diversity. We used comparative genomics in combination with analysis of existing transcriptomic data to identify three novel putative RNA structures associated with the S15 coding region in microbial genomes. These structures are completely distinct from those previously published and encompass potential regulatory regions including ribosome-binding sites. To validate the biological relevance of our findings, we demonstrate that an example of the Alphaproteobacterial RNA from Rhizobium radiobacter specifically interacts with S15 in vitro, and allows in vivo regulation of gene expression in an E. coli reporter system. In addition, structural probing and nuclease protection assays confirm the predicted secondary structure and indicate nucleotides required for protein interaction. This work illustrates the importance of integrating comparative genomic and transcriptomic approaches during de novo ncRNA identification and reveals a diversity of distinct natural RNA regulators that support analogous biological functions. Furthermore, this work indicates that many additional uncharacterized RNA regulators likely exist within bacterial genomes and that the plasticity of RNA structure allows unique, and likely independently derived, solutions to the same biological problem.
    BMC Genomics 08/2014; 15(1):657. DOI:10.1186/1471-2164-15-657 · 3.99 Impact Factor
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    • "Moreover, the subsequent further enrichment of D. shibae mRNA using the MICROBExpress kit was also not effective, with 90% and 93.8% rRNA reads remaining in replicate 1 and replicate 2, respectively. A recent study by Giannoukos et al. (2012) comparing 5 different rRNA removal methods showed a low efficiency of the MICROBExpress kit and a very high efficiency of the Ribo-Zero kit from Epicenter in 3 tested bacteria, among which Rhodobacter sphaeroides is very closely related to D. shibae. Thus, we tested the Ribo-Zero magnetic kit for gram-negative bacteria on a pure culture of D. shibae culture. "
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    Frontiers in Microbiology 06/2014; 5:311. DOI:10.3389/fmicb.2014.00311 · 3.99 Impact Factor
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