Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies
| Autor: | Elmar Pruesse, Anna Klindworth, Frank Oliver Glöckner, Christian Quast, Matthias Horn, Jörg Peplies, Timmy Schweer |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Sequence analysis
archaea polymerase chain reaction Biology amplification DNA sequencing 03 medical and health sciences Genetics Computer Simulation genes bacteria DNA Primers 030304 developmental biology 0303 health sciences 030306 microbiology datasets Genetic Variation High-Throughput Nucleotide Sequencing Genes rRNA Biodiversity Sequence Analysis DNA Ribosomal RNA Amplicon 16S ribosomal RNA Amplicon Size dna ribosomal Metagenomics rna ribosomal 16s Methods Online Metagenome Primer (molecular biology) mismatch |
| Zdroj: | Nucleic Acids Research (London) Nucleic Acids Research |
| Popis: | 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, epsilon 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. |
| Databáze: | OpenAIRE |
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