Artículos de revistas
Multiplex Amplicon Sequencing For Microbe Identification In Community-based Culture Collections
Registro en:
Scientific Reports. Nature Publishing Group, v. 6, p. , 2016.
2045-2322
WOS:000379396300001
10.1038/srep29543
Autor
Leite Armanhi
Jaderson Silveira; Correa de Souza
Rafael Soares; de Araujo
Laura Migliorini; Okura
Vagner Katsumi; Mieczkowski
Piotr; Imperial
Juan; Arruda
Paulo
Institución
Resumen
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. 6 Repsol Repsol Sinopec