Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing

University of California Davis, United States of America
PLoS Genetics (Impact Factor: 7.53). 12/2008; 4(11):e1000255. DOI: 10.1371/journal.pgen.1000255
Source: PubMed


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.

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    • "In some studies on the endophytes using culture-independent approaches, Gammaproteobacteria dominated, with the genus Pseudomonas comprising 42% of the total isolate collection (Moore et al., 2006). It has been previously observed that in many cases, Pseudomonas are abundant in both the soil environment (Samish et al., 1963; Spiers et al., 2000) and inside the plant (Gardner et al., 1982; Tanprasert and Reed, 1997; Rademaker et al., 1998; Huse et al., 2008). It has been suggested that endophytic bacteria colonize plants primarily through the root network via natural and artificial wound sites, root hairs and at epidermal junctions (Sprent and de Faria, 1988; Pan et al., 1997). "
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    • "While the use of the SSU rRNA gene as a conserved phylogenetic marker has advanced our understanding of microbial communities, its application can be limited when resolving phylogenetic relationships of early divergent microorganisms such as cyanobacteria (Sánchez- Baracaldo et al., 2005) because multiple substitutions at individual loci over long periods of time can lead to false tree topologies when single genes are used, regardless of the number of sequences (Philippe et al., 2011). Furthermore, the ability of short reads to accurately resolve phylogenetic relations has been called into question (Huse et al., 2008; Liu et al., 2009; Youssef et al., 2009) and reliance upon fragmented SSU rRNA gene sequences from environmental samples alone can be problematic. Previous studies of cold filamentous cyanobacteria have treated paraphyletic groups as monophyletic (Casamatta et al., 2005; Strunecký et al., 2010) resulting in misleading interpretations of their evolutionary histories. "
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    • "due to the relatively lower resolution of these techniques , the diversity of plant endophytic bacteria is far from being understood . The emergence of next - generation sequencing technology allows sequence efforts that are orders of magnitude greater than ever before and therefore have opened a new frontier in microbial diversity determination ( Huse et al . , 2008 ; Hollister et al . , 2010 ) . This technique has been used to evaluate the diversity of endophytic bacterial communities from the roots of rice ( Zhang et al . , 2013 ) and potato ( Manter et al . , 2010 ) , and the leaves of rice ( Ren et al . , 2015 ) and commercial salad ( e . g . , baby spinach and lettuce ; Jackson et al . , 2013 "
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