Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing

Abstract

Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy by creating gene trees de novo that include all experimental sequences and certain reference taxa, we compare the hypervariable region tags to an extensive database of rRNA sequences and assign taxonomy based on the best match in a Global Alignment for Sequence Taxonomy (GAST) process. The resulting taxonomic census provides information on both composition and diversity of the microbial community. To determine the effectiveness of using only hypervariable region tags for assessing microbial community membership, we compared the taxonomy assigned to the V3 and V6 hypervariable regions with the taxonomy assigned to full-length SSU rRNA sequences isolated from both the human gut and a deep-sea hydrothermal vent. The hypervariable region tags and full-length rRNA sequences provided equivalent taxonomy and measures of relative abundance of microbial communities, even for tags up to 15% divergent from their nearest reference match. The greater sampling depth per dollar afforded by massively parallel pyrosequencing reveals many more members of the “rare biosphere” than does capillary sequencing of the full-length gene. In addition, tag sequencing eliminates cloning bias and the sequences are short enough to be completely sequenced in a single read, maximizing the number of organisms sampled in a run while minimizing chimera formation. This technique allows the cost-effective exploration of changes in microbial community structure, including the rare biosphere, over space and time and can be applied immediately to initiatives, such as the Human Microbiome Project.

Effect of an extruded animal protein‐free diet on fecal microbiota of dogs with food‐responsive enteropathy

“…In dogs with acute diarrhea, after perturbation the microbiota seemed to return to the starting condition 45. Administration of antibiotics is a strong driver to shift GI microbial composition 16, 46, 47. In 1 study, treatment with diet, with or without a probiotic strain ( Enterococcus faecium ), lead to a significantly increased richness of the fecal bacterial microbiome in dogs with FRE, similar to our results 48…”

Biogeography and ecology of the rare and abundant microbial lineages in deep-sea hydrothermal vents

“…The average 140 temperature of the sample, as determined from its silica and magnesium concentrations, 141 was approximately 125˚C (Anderson et al 2011). This sample most likely represents a 142 analyses of each sample using the GAST process in VAMPS (Huse et al 2008). We 188 further screened, filtered and trimmed all sequences as a batch set using mothur (Schloss 189 et al 2009).…”

The 16S rRNA gene in the study of marine microbial communities

“…In a characterization of microbial communities in wastewater, the information obtained with a fragment containing the V1 and V2 regions proved insufficient to detect organisms belonging to the phyla Verrucomicrobia, Planctomycetes, and Chlamydiae (Cai et al 2013). In another study analyzing samples from the human gut and deep-sea vents, differences were observed in the diversity found for the 16S rRNA regions used: 42 taxa were detected in the V3 region and only 26 in the V6 region (Huse et al 2008). excelente marcador molecular y se han planteado nuevas estrategias de estudio, aprovechando las bondades de las nuevas técnicas genómicas (Savolainen et al 2005, Tanabe y Toju 2013.…”

“…En la caracterización de comunidades microbianas de aguas residuales, la información obtenida con un fragmento que contenía a las regiones V1 y V2 no fue suficiente para detectar organismos de los filos Verrucomicrobia, Planctomycetes y Chlamydiae (Cai et al 2013). En otro estudio, Huse et al (2008) analizaron muestras tan distintas como de intestino humano y chimeneas submarinas y demostraron que cada región del ARNr 16S proporciona diferentes valores de diversidad microbiana: mientras que con el uso de la región V3 registraron 42 taxones, con la V6 solamente encontraron 26. Pese a estos inconvenientes, el uso del ARNr 16S como marcador sigue siendo la herramienta más fuerte para el entendimiento de las comunidades bacterianas de todos los ambientes estudiados.…”