title: A comprehensive metatranscriptome analysis pipeline and its validation using human small intestine microbiota datasets.
abstract: BACKGROUND
  Next generation sequencing (NGS) technologies can be applied in complex microbial ecosystems for metatranscriptome analysis by employing direct cDNA sequencing, which is known as RNA sequencing (RNA-seq). RNA-seq generates large datasets of great complexity, the comprehensive interpretation of which requires a reliable bioinformatic pipeline. In this study, we focus on the development of such a metatranscriptome pipeline, which we validate using Illumina RNA-seq datasets derived from the small intestine microbiota of two individuals with an ileostomy.
  
  
  RESULTS
  The metatranscriptome pipeline developed here enabled effective removal of rRNA derived sequences, followed by confident assignment of the predicted function and taxonomic origin of the mRNA reads. Phylogenetic analysis of the small intestine metatranscriptome datasets revealed a strong similarity with the community composition profiles obtained from 16S rDNA and rRNA pyrosequencing, indicating considerable congruency between community composition (rDNA), and the taxonomic distribution of overall (rRNA) and specific (mRNA) activity among its microbial members. Reproducibility of the metatranscriptome sequencing approach was established by independent duplicate experiments. In addition, comparison of metatranscriptome analysis employing single- or paired-end sequencing methods indicated that the latter approach does not provide improved functional or phylogenetic insights. Metatranscriptome functional-mapping allowed the analysis of global, and genus specific activity of the microbiota, and illustrated the potential of these approaches to unravel syntrophic interactions in microbial ecosystems.
  
  
  CONCLUSIONS
  A reliable pipeline for metatransciptome data analysis was developed and evaluated using RNA-seq datasets obtained for the human small intestine microbiota. The set-up of the pipeline is very generic and can be applied for (bacterial) metatranscriptome analysis in any chosen niche.
authors.affiliation: TI Food and Nutrition (TIFN), P,O, Box 557, 6700 AN, Wageningen, The Netherlands.
authors.forename: Milkha M
authors.initials: MM
authors.lastname: Leimena
authors.forename: Javier
authors.initials: J
authors.lastname: Ramiro-Garcia
authors.forename: Mark
authors.initials: M
authors.lastname: Davids
authors.forename: Bartholomeus
authors.initials: B
authors.lastname: van den Bogert
authors.forename: Hauke
authors.initials: H
authors.lastname: Smidt
authors.forename: Eddy J
authors.initials: EJ
authors.lastname: Smid
authors.forename: Jos
authors.initials: J
authors.lastname: Boekhorst
authors.forename: Erwin G
authors.initials: EG
authors.lastname: Zoetendal
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Schaap
authors.forename: Michiel
authors.initials: M
authors.lastname: Kleerebezem
mesh_terms: D000368:Aged; D019295:Computational Biology / Q000379:methods* / Q000592:standards; D030541:Databases, Genetic; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D006801:Humans; D007421:Intestine, Small / Q000382:microbiology*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D054892:Metagenome / Q000235:genetics*; D008875:Middle Aged; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics; D012015:Reference Standards; D017423:Sequence Analysis, RNA
chemical_list: D012333:RNA, Messenger
references.citation: FEMS Microbiol Ecol. 2013 Aug;85(2):376-88
references.pmid: 23614882
references.citation: Proc Natl Acad Sci U S A. 2010 Apr 20;107(16):7503-8
references.pmid: 20363958
references.citation: Biotechniques. 1996 Nov;21(5):934-6, 938-9
references.pmid: 8922637
references.citation: Nucleic Acids Res. 2011 Jul;39(Web Server issue):W412-5
references.pmid: 21546551
references.citation: Nat Methods. 2008 Mar;5(3):235-7
references.pmid: 18264105
references.citation: ISME J. 2012 Feb;6(2):248-58
references.pmid: 21776033
references.citation: Nat Methods. 2008 Jul;5(7):621-8
references.pmid: 18516045
references.citation: Genome Biol. 2007;8(11):R238
references.pmid: 17996061
references.citation: Nucleic Acids Res. 2009 Jan;37(Database issue):D489-93
references.pmid: 18948294
references.citation: Brief Bioinform. 2013 Nov;14(6):671-83
references.pmid: 22988256
references.citation: Nature. 2013 Jan 3;493(7430):45-50
references.pmid: 23222524
references.citation: Appl Environ Microbiol. 2005 Aug;71(8):4679-89
references.pmid: 16085863
references.citation: Appl Environ Microbiol. 2012 Jun;78(12):4141-8
references.pmid: 22492454
references.citation: Bioinformatics. 2012 Dec 15;28(24):3211-7
references.pmid: 23071270
references.citation: Nat Rev Genet. 2010 Jan;11(1):9-16
references.pmid: 19935729
references.citation: J Biotechnol. 2009 Jun 1;142(1):91-5
references.pmid: 19480952
references.citation: PLoS One. 2008 Aug 22;3(8):e3042
references.pmid: 18725995
references.citation: Bioinformatics. 2010 Apr 15;26(8):996-9
references.pmid: 20231229
references.citation: Genome Res. 2011 Mar;21(3):494-504
references.pmid: 21212162
references.citation: Nature. 2010 Mar 4;464(7285):59-65
references.pmid: 20203603
references.citation: Syst Appl Microbiol. 1999 Sep;22(3):434-44
references.pmid: 10553296
references.citation: ISME J. 2012 Jul;6(7):1415-26
references.pmid: 22258098
references.citation: PLoS One. 2011 Mar 08;6(3):e17447
references.pmid: 21408168
references.citation: Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3805-10
references.pmid: 18316740
references.citation: Nucleic Acids Res. 2007 Jul;35(Web Server issue):W182-5
references.pmid: 17526522
references.citation: Environ Microbiol. 2010 Dec;12(12):3213-27
references.pmid: 20626454
references.citation: Environ Microbiol. 2009 Apr;11(4):914-26
references.pmid: 19128319
references.citation: Nucleic Acids Res. 2007;35(21):7188-96
references.pmid: 17947321
references.citation: Appl Environ Microbiol. 2000 Nov;66(11):4605-14
references.pmid: 11055900
references.citation: PLoS One. 2011;6(7):e22448
references.pmid: 21829462
references.citation: Genome Biol. 2012;13(3):R23
references.pmid: 22455878
references.citation: J Biotechnol. 2007 Jan 20;127(4):549-59
references.pmid: 16945445
references.citation: Genome Res. 2008 Sep;18(9):1509-17
references.pmid: 18550803
references.citation: Nat Protoc. 2006;1(2):954-9
references.pmid: 17406329
references.citation: Appl Environ Microbiol. 2000 Apr;66(4):1328-33
references.pmid: 10742207
references.citation: Methods Mol Biol. 2011;733:195-205
references.pmid: 21431772
references.citation: Future Microbiol. 2007 Jun;2(3):285-95
references.pmid: 17661703
references.citation: ISME J. 2012 Jul;6(7):1273-6
references.pmid: 22170427
references.citation: J Clin Microbiol. 2010 Mar;48(3):900-7
references.pmid: 20053859
references.citation: Methods. 2009 Jul;48(3):240-8
references.pmid: 19275939
references.citation: Proc Natl Acad Sci U S A. 2009 Mar 10;106(10):3976-81
references.pmid: 19234113
references.citation: J Bacteriol. 2009 May;191(10):3203-11
references.pmid: 19304856
references.citation: Genome Biol. 2010;11(3):108
references.pmid: 20236470
references.citation: Dig Dis. 2011;29(6):525-30
references.pmid: 22179207
references.citation: PLoS One. 2012;7(4):e36009
references.pmid: 22558305
references.citation: Nucleic Acids Res. 2000 Jan 1;28(1):33-6
references.pmid: 10592175
references.citation: Nucleic Acids Res. 2009 Jan;37(Database issue):D141-5
references.pmid: 19004872
references.citation: Nucleic Acids Res. 2011 Jul;39(13):e90
references.pmid: 21576222
references.citation: Nucleic Acids Res. 2000 Jan 1;28(1):27-30
references.pmid: 10592173
references.citation: FEMS Microbiol Ecol. 2011 Jan;75(1):2-16
references.pmid: 20662931
references.citation: Nat Methods. 2010 May;7(5):335-6
references.pmid: 20383131
references.citation: PLoS One. 2008 Jun 25;3(6):e2527
references.pmid: 18575584
references.citation: Bioinformatics. 2011 Mar 15;27(6):867-9
references.pmid: 21233166
references.citation: Appl Environ Microbiol. 2011 Mar;77(6):2071-80
references.pmid: 21257804