Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies

By Yong Wang, Pei-Yuan Qian. PLoS ONE, 2009

1,159 citations recorded by the source corpus.

Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519–539, E969–983, E1063–1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies.

Subfields: Ecology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/efdba25b13c217b52790c95a5a7d23b4537fc70e

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Citations in the corpus

The under-recognized dominance of Verrucomicrobia in soil bacterial communities (2011, background): “It is likely that verrucomicrobial abundances have been underestimated in many studies, as a number of commonly used PCR primers targeting 16S rRNA are biased against members of the Verrucomicrobia (see Fig. S1, and Tables S4 and S5; see also: Andersson et al., 2008; Hamady and Knight, 2009; Wang an”
454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants (2011, background): “First, the primers applied may generate 16S rRNA gene fragments with limited efficiency (Hong et al., 2009; Wang and Qian, 2009), although the primer selection bias is the same for all samples and would not significantly affect the overall comparison of these sludges.”
Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies (2012, background): “%) is misspelled in Wang and Qian (15).”
Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions (2010, positive): “In a separate study, Wang and colleagues highlighted the lack of coverage and scope estimates for known 16S rRNA primers, and in response generated a comprehensive list of tested primers, along with recommendations of a few with particularly high coverage and universal properties (20).”