title: Functional Profiling of Unfamiliar Microbial Communities Using a Validated De Novo Assembly Metatranscriptome Pipeline.
abstract: BACKGROUND
  Metatranscriptomic landscapes can provide insights in functional relationships within natural microbial communities. Analysis of complex metatranscriptome datasets of these communities poses a considerable bioinformatic challenge since they are non-restricted with a varying number of participating strains and species. For RNA-Seq data a standard approach is to align the generated reads to a set of closely related reference genomes. This only works well for microbial communities for which a near complete catalogue of reference genomes is available at a small evolutionary distance. In this study, we focus on the design of a validated de novo metatranscriptome assembly pipeline for single-end Illumina RNA-Seq data to obtain functional and taxonomic profiles of murine microbial communities.
  
  
  RESULTS
  The here developed de novo assembly metatranscriptome pipeline combined rRNA removal, IDBA-UD assembler, functional annotation and taxonomic classification. Different assemblers were tested and validated using RNA-Seq data from an in silico generated mock community and in vivo RNA-Seq data from a restricted microbial community taken from a mouse model colonized with Altered Schaedler Flora (ASF). Precision and recall of resulting gene expression, functional and taxonomic profiles were compared to those obtained with a standard alignment method. The validated pipeline was subsequently used to generate expression profiles from non-restricted cecal communities of four C57BL/6J mice fed on a high-fat high-protein diet spiked with an RNA-Seq data set from a well-characterized human sample. The spike in control was used to estimate precision and recall at assembly, functional and taxonomic level of non-restricted communities.
  
  
  CONCLUSIONS
  A generic de novo assembly pipeline for metatranscriptome data analysis was designed for microbial ecosystems, which can be applied for microbial metatranscriptome analysis in any chosen niche.
authors.affiliation: Laboratory of Systems and Synthetic Biology, Wageningen University, Dreijenplein 10, Wageningen, The Netherlands.|Netherlands Consortium for Systems Biology, TI Food and Nutrition, Wageningen, The Netherlands.
authors.forename: Mark
authors.initials: M
authors.lastname: Davids
authors.affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, Wageningen, The Netherlands.|Netherlands Consortium for Systems Biology, TI Food and Nutrition, Wageningen, The Netherlands.
authors.forename: Floor
authors.initials: F
authors.lastname: Hugenholtz
authors.affiliation: Laboratory of Systems and Synthetic Biology, Wageningen University, Dreijenplein 10, Wageningen, The Netherlands.|Netherlands Consortium for Systems Biology, TI Food and Nutrition, Wageningen, The Netherlands.
authors.forename: Vitor
authors.initials: V
authors.lastname: Martins dos Santos
authors.affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, Wageningen, The Netherlands.|Netherlands Consortium for Systems Biology, TI Food and Nutrition, Wageningen, The Netherlands.
authors.forename: Hauke
authors.initials: H
authors.lastname: Smidt
authors.affiliation: Host-Microbe Interactomics Group, Wageningen University, Wageningen, The Netherlands.|Netherlands Consortium for Systems Biology, TI Food and Nutrition, Wageningen, The Netherlands.
authors.forename: Michiel
authors.initials: M
authors.lastname: Kleerebezem
authors.affiliation: Laboratory of Systems and Synthetic Biology, Wageningen University, Dreijenplein 10, Wageningen, The Netherlands.|Netherlands Consortium for Systems Biology, TI Food and Nutrition, Wageningen, The Netherlands.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Schaap
mesh_terms: D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism; D002432:Cecum / Q000382:microbiology; D030541:Databases, Genetic; D020869:Gene Expression Profiling; D006801:Humans; D007422:Intestines / Q000382:microbiology; D053858:Metabolic Networks and Pathways / Q000235:genetics; D056186:Metagenomics / Q000379:methods*; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D013045:Species Specificity; D059467:Transcriptome / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins