title: Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures.
abstract: 16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current limitations and drawbacks of 16S rRNA gene profiling. Although sample handling, DNA extraction methods and the choice of universal 16S rRNA gene PCR primers are well known factors that could seriously affect the final results of microbiota profiling studies, inevitable amplification artifacts, such as chimera formation and PCR competition, are seldom appreciated. Here we report on a novel micelle based amplification strategy, which overcomes these limitations via the clonal amplification of targeted DNA molecules. Our results show that micelle PCR drastically reduces chimera formation by a factor of 38 (1.5% vs. 56.9%) compared with traditional PCR, resulting in improved microbial diversity estimates. In addition, compartmentalization during micelle PCR prevents PCR competition due to unequal amplification rates of different 16S template molecules, generating robust and accurate 16S microbiota profiles required for comparative studies (e.g. longitudinal surveys).
authors.affiliation: Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Centre, Wytemaweg 80, 3015 CN, Rotterdam, The Netherlands.
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Boers
authors.affiliation: Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Centre, Wytemaweg 80, 3015 CN, Rotterdam, The Netherlands.
authors.forename: John P
authors.initials: JP
authors.lastname: Hays
authors.affiliation: Department of Molecular Biology, Regional Laboratory of Public Health Kennemerland, Boerhaavelaan 26, 2035 RC, Haarlem, The Netherlands.
authors.forename: Ruud
authors.initials: R
authors.lastname: Jansen
mesh_terms: D059014:High-Throughput Nucleotide Sequencing; D054892:Metagenome; D056186:Metagenomics / Q000379:methods; D064307:Microbiota; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D012336:RNA, Ribosomal, 16S