a comparison of rpob and 16s rrna as markers in pyrosequencing studies of bacterial diversity比较rpob和16 s rrna焦磷酸测序研究的细菌多样性的标记.pdfVIP

A Comparison of rpoB and 16S rRNA as Markers in Pyrosequencing Studies of Bacterial Diversity 1 .¤ 2. 1 1 1,3 Michiel Vos * , Christopher Quince , Agata S. Pijl , Mattias de Hollander , George A. Kowalchuk 1 Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, The Netherlands, 2 Department of Civil Engineering, University of Glasgow, Glasgow, United Kingdom, 3 Institute of Ecological Science, Free University of Amsterdam, Amsterdam, The Netherlands Abstract Background: The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria. Methodology/Principal Findings: Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genet