Technical challenges in metatranscriptomic studies applied to the bacterial communities of freshwater ecosystems
2014
Pascault, Noémie | Loux, Valentin | Derozier, Sandra | Martin, Véronique | Debroas, Didier | Maloufi, Selma | Humbert, Jean-François | Leloup, Julie | Microbiologie du Sol et de l'Environnement (MSE) ; Institut National de la Recherche Agronomique (INRA)-Université de Bourgogne (UB) | INRA Transfert | Unité Mathématique Informatique et Génome (MIG) ; Institut National de la Recherche Agronomique (INRA) | Centre de Robotique (CAOR) ; Mines Paris - PSL (École nationale supérieure des mines de Paris) ; Université Paris Sciences et Lettres (PSL)-Université Paris Sciences et Lettres (PSL) | Laboratoire Microorganismes : Génome et Environnement (LMGE) ; Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Université d'Auvergne - Clermont-Ferrand I (UdA)-Centre National de la Recherche Scientifique (CNRS) | Molécules de Communication et Adaptation des Micro-organismes (MCAM) ; Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS) | Laboratoire Image, Ville, Environnement (LIVE) ; Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS) | Institut d'écologie et des sciences de l'environnement de Paris (iEES) ; Institut National de la Recherche Agronomique (INRA)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Centre National de la Recherche Scientifique (CNRS)
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Mostrar más [+] Menos [-]Inglés. Metatranscriptome analysis relates to the transcriptome of microbial communities directly sampled in the environment. Accessing the mRNA pool in natural bacterial communities presents some technical challenges such as the RNA extraction, rRNA depletion, and the choice of the high-throughput sequencing technique. The lack of technical details in scientific articles is a major problem to correctly obtained mRNA from a microbial community and thus the corresponding sequencing data. In our study, we present the methodological procedure that was developed in order to access to the metatranscriptome of the microbial communities during two cyanobacterial blooms successively occurring in a freshwater eutrophic lake. Each procedure step was detailed and discussed with regard to the choices and difficulties encountered and to the recent literature. Finally, the two major limits for metatranscriptomic approaches targeting bacterial communities from natural environments were (i) the removal of rRNA in order to increase the putative mRNA reads number after sequencing, and (ii) for most of the bacterial communities living in natural environments, the lack of reference genomes in databases that leads to the non-assignation of numerous reads. Once these challenges overcome, we managed to access putative mRNA of dominant species, i.e. cyanobacteria (from 6 to 72 % of mRNA assigned), and of the surrounding bacteria (from 1 to 5 % of mRNA assigned).
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