title: Comparison of library preparation methods reveals their impact on interpretation of metatranscriptomic data.
abstract: BACKGROUND
  Metatranscriptomics is rapidly expanding our knowledge of gene expression patterns and pathway dynamics in natural microbial communities. However, to cope with the challenges of environmental sampling, various rRNA removal and cDNA synthesis methods have been applied in published microbial metatranscriptomic studies, making comparisons arduous. Whereas efficiency and biases introduced by rRNA removal methods have been relatively well explored, the impact of cDNA synthesis and library preparation on transcript abundance remains poorly characterized. The evaluation of potential biases introduced at this step is challenging for metatranscriptomic samples, where data analyses are complex, for example because of the lack of reference genomes.
  
  
  RESULTS
  Herein, we tested four cDNA synthesis and Illumina library preparation protocols on a simplified mixture of total RNA extracted from four bacterial species. In parallel, RNA from each microbe was tested individually. cDNA synthesis was performed on rRNA depleted samples using the TruSeq Stranded Total RNA Library Preparation, the SMARTer Stranded RNA-Seq, or the Ovation RNA-Seq V2 System. A fourth experiment was made directly from total RNA using the Encore Complete Prokaryotic RNA-Seq. The obtained sequencing data were analyzed for: library complexity and reproducibility; rRNA removal efficiency and bias; the number of genes detected; coverage uniformity; and the impact of protocols on expression biases. Significant variations, especially in organism representation and gene expression patterns, were observed among the four methods. TruSeq generally performed best, but is limited by its requirement of hundreds of nanograms of total RNA. The SMARTer method appears the best solution for smaller amounts of input RNA. For very low amounts of RNA, the Ovation System provides the only option; however, the observed biases emphasized its limitations for quantitative analyses.
  
  
  CONCLUSIONS
  cDNA and library preparation methods may affect the outcome and interpretation of metatranscriptomic data. The most appropriate method should be chosen based on the available quantity of input RNA and the quantitative or non-quantitative objectives of the study. When low amounts of RNA are available, as in most metatranscriptomic studies, the SMARTer method seems to be the best compromise to obtain reliable results. This study emphasized the difficulty in comparing metatranscriptomic studies performed using different methods.
authors.affiliation: CEA-Institut de Génomique, Genoscope, Centre National de Séquençage, 2 rue Gaston Crémieux, CP5706, F-91057 Evry Cedex, France. aalberti@genoscope.cns.fr.
authors.forename: Adriana
authors.initials: A
authors.lastname: Alberti
authors.forename: Caroline
authors.initials: C
authors.lastname: Belser
authors.forename: Stéfan
authors.initials: S
authors.lastname: Engelen
authors.forename: Laurie
authors.initials: L
authors.lastname: Bertrand
authors.forename: Céline
authors.initials: C
authors.lastname: Orvain
authors.forename: Laura
authors.initials: L
authors.lastname: Brinas
authors.forename: Corinne
authors.initials: C
authors.lastname: Cruaud
authors.forename: Laurène
authors.initials: L
authors.lastname: Giraut
authors.forename: Corinne
authors.initials: C
authors.lastname: Da Silva
authors.forename: Cyril
authors.initials: C
authors.lastname: Firmo
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Aury
authors.forename: Patrick
authors.initials: P
authors.lastname: Wincker
mesh_terms: D001419:Bacteria / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D013223:Statistics as Topic / Q000379:methods*; D059467:Transcriptome / Q000235:genetics*
chemical_list: D012333:RNA, Messenger