Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies
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Open Access
- 9 October 2009
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 4 (10), e7401
- https://doi.org/10.1371/journal.pone.0007401
Abstract
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.Keywords
This publication has 27 references indexed in Scilit:
- Human gut microbiota in obesity and after gastric bypassProceedings of the National Academy of Sciences of the United States of America, 2009
- Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag SequencingPLoS Genetics, 2008
- The Ribosomal Database Project: improved alignments and new tools for rRNA analysisNucleic Acids Research, 2008
- Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencersNucleic Acids Research, 2008
- Specificity and sensitivity of eubacterial primers utilized for molecular profiling of bacteria within complex microbial ecosystemsJournal of Microbiological Methods, 2007
- Short pyrosequencing reads suffice for accurate microbial community analysisNucleic Acids Research, 2007
- Pyrosequencing enumerates and contrasts soil microbial diversityThe ISME Journal, 2007
- Classification, Identification And Subtyping Of Bacteria Based On Pyrosequencing And Signature Matching Of 16s rDNA FragmentsAPMIS, 2007
- Microbial diversity in the deep sea and the underexplored “rare biosphere”Proceedings of the National Academy of Sciences of the United States of America, 2006
- Mitochondrial EvolutionScience, 1999