%0 Journal Article
%T The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons
%A Ingo C. Starke
%A Wilfried Vahjen
%A Robert Pieper
%A J¨¹rgen Zentek
%J Molecular Biology International
%D 2014
%I Hindawi Publishing Corporation
%R 10.1155/2014/548683
%X In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5 sequences were used for analysis after processing for read length (150£¿bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis. 1. Introduction Molecular tools such as the recently introduced method of massively parallel sequencing (deep sequencing) [1, 2] greatly facilitate the study of complex bacterial communities and provide deep insights into their compositions [3¨C5]. Combined with the technique of barcoded PCR amplicons, deep sequencing methods are able to process many samples at a relatively low cost per sequence [6, 7]. Deep sequencing is, therefore, a promising tool for examining the influence of nutritional and other factors on intestinal microbial communities and functionalities. However, as with any new technology, pitfalls exist. For barcoded PCR amplicon sequencing studies, nucleic acids must be extracted and the resulting DNA extract should ideally represent the entire bacterial diversity in a given habitat. Furthermore, barcoding requires a PCR step, which depends on primers that should ideally cover the complete bacterial diversity. Finally, the evaluation of sequence reads is based on databases, most of which are not yet suited for massive sequence inputs [8] and sequence quality is often found to be suboptimal [9, 10]. In regard to DNA extraction from complex samples, a multitude of studies have reported that any given nucleic acid extraction method is biased towards certain bacterial groups [11¨C13]. Complex samples such as
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