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Genome coverage and sequence fidelity of φ29 polymerase-based multiple strand displacement whole genome amplification

Nucleic Acids Research
Oxford University Press
Publication Date
DOI: 10.1093/nar/gnh069
  • Nar Methods Online
  • Biology
  • Computer Science


OP-NARE120488 1..11 Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies Anna Klindworth1,2, Elmar Pruesse1,2, Timmy Schweer1, Jo¨rg Peplies3, Christian Quast1, Matthias Horn4 and Frank Oliver Glo¨ckner1,2,* 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 Received December 12, 2011; Accepted July 31, 2012 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, �1000bp) 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 464bp, 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 select- ing primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies. INTRODUCTION Understanding microbial diversity has been the ambition of scientists for decades. Because diversity analysis by cultivation is problematic for a significant fraction of Bacteria and Archaea, culture-independent surveys

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