title: Novel micelle PCR-based method for accurate, sensitive and quantitative microbiota profiling.
abstract: In the last decade, many researchers have embraced 16S rRNA gene sequencing techniques, which has led to a wealth of publications and documented differences in the composition of microbial communities derived from many different ecosystems. However, comparison between different microbiota studies is currently very difficult due to the lack of a standardized 16S rRNA gene sequencing protocol. Here we report on a novel approach employing micelle PCR (micPCR) in combination with an internal calibrator that allows for standardization of microbiota profiles via their absolute abundances. The addition of an internal calibrator allows the researcher to express the resulting operational taxonomic units (OTUs) as a measure of 16S rRNA gene copies by correcting the number of sequences of each individual OTU in a sample for efficiency differences in the NGS process. Additionally, accurate quantification of OTUs obtained from negative extraction control samples allows for the subtraction of contaminating bacterial DNA derived from the laboratory environment or chemicals/reagents used. Using equimolar synthetic microbial community samples and low biomass clinical samples, we demonstrate that the calibrated micPCR/NGS methodology possess a much higher precision and a lower limit of detection compared with traditional PCR/NGS, resulting in more accurate microbiota profiles suitable for multi-study comparison.
authors.affiliation: Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Centre Rotterdam (Erasmus MC), 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 Rotterdam (Erasmus MC), 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, Haarlem, The Netherlands.
authors.forename: Ruud
authors.initials: R
authors.lastname: Jansen
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics; D056186:Metagenomics / Q000379:methods* / Q000592:standards; D064307:Microbiota; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S