title: Multiplex amplicon sequencing for microbe identification in community-based culture collections.
abstract: Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas (UNICAMP), 13083-875, Campinas, SP, Brazil.
authors.forename: Jaderson Silveira Leite
authors.initials: JS
authors.lastname: Armanhi
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas (UNICAMP), 13083-875, Campinas, SP, Brazil.
authors.forename: Rafael Soares Correa
authors.initials: RS
authors.lastname: de Souza
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas (UNICAMP), 13083-875, Campinas, SP, Brazil.
authors.forename: Laura Migliorini
authors.initials: LM
authors.lastname: de Araújo
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas (UNICAMP), 13083-875, Campinas, SP, Brazil.
authors.forename: Vagner Katsumi
authors.initials: VK
authors.lastname: Okura
authors.affiliation: Department of Genetics, University of North Carolina, Chapel Hill, North Carolina, USA.
authors.forename: Piotr
authors.initials: P
authors.lastname: Mieczkowski
authors.affiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, 28223, Pozuelo de Alarcón, (Madrid), Spain.|Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Imperial
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas (UNICAMP), 13083-875, Campinas, SP, Brazil.|Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), 13083-970, Campinas, SP, Brazil.
authors.forename: Paulo
authors.initials: P
authors.lastname: Arruda
mesh_terms: D046508:Culture Techniques; D004269:DNA, Bacterial; D005827:Genetics, Microbial; D056186:Metagenomics / Q000379:methods*; D008828:Microbiological Techniques; D064307:Microbiota; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S; D031786:Saccharum / Q000382:microbiology; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S