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Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies

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      Abstract

      16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of ‘best available’ primer pairs for Bacteria and Archaea for three amplicon size classes (100–400, 400–1000, ≥1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.

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      Search and clustering orders of magnitude faster than BLAST.

       Robert Edgar (2010)
      Biological sequence data is accumulating rapidly, motivating the development of improved high-throughput methods for sequence classification. UBLAST and USEARCH are new algorithms enabling sensitive local and global search of large sequence databases at exceptionally high speeds. They are often orders of magnitude faster than BLAST in practical applications, though sensitivity to distant protein relationships is lower. UCLUST is a new clustering method that exploits USEARCH to assign sequences to clusters. UCLUST offers several advantages over the widely used program CD-HIT, including higher speed, lower memory use, improved sensitivity, clustering at lower identities and classification of much larger datasets. Binaries are available at no charge for non-commercial use at http://www.drive5.com/usearch.
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        Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB.

        A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages in the Archaea and Bacteria.
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          Genome sequencing in microfabricated high-density picolitre reactors.

          The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.
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            Author and article information

            Affiliations
            1Max Planck Institute for Marine Microbiology, Microbial Genomics and Bioinformatics Research Group, Celsiusstr.1, 28359 Bremen, 2Jacobs University Bremen, School of Engineering and Sciences, Campusring 1, 28759 Bremen, 3Ribocon GmbH, D-28359 Bremen, Germany and 4Department of Microbial Ecology, University of Vienna, Althanstr. 14, 1090 Vienna, Austria
            Author notes
            *To whom correspondence should be addressed. Tel: +49 421 2028970; Fax: +49 421 2028580; Email: fog@ 123456mpi-bremen.de
            Journal
            Nucleic Acids Res
            Nucleic Acids Res
            nar
            nar
            Nucleic Acids Research
            Oxford University Press
            0305-1048
            1362-4962
            January 2013
            28 August 2012
            28 August 2012
            : 41
            : 1
            : e1
            22933715
            3592464
            10.1093/nar/gks808
            gks808
            © The Author(s) 2012. Published by Oxford University Press.

            This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.

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            Pages: 11
            Categories
            Methods Online
            Custom metadata
            7 January 2013

            Genetics

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