Abstract

Pyrosequencing of 16S rRNA gene amplicons on the 454 FLX Titanium platform has been widely used to analyze microbiomes in various environments. However, different results may stem from variations among sequencing runs or among sequencing facilities. This study aimed to evaluate these variations between different pyrosequencing runs by sequencing 16S rRNA gene amplicon libraries generated from three sets of rumen samples twice each on the 454 FLX Titanium system at two independent sequencing facilities. Similar relative abundances were found for predominant taxa represented by large numbers of sequence reads but not for minor taxa represented by small numbers of sequence reads. The two sequencing facilities revealed different bac-terial profiles with respect to both predominant taxa and minor taxa, including the most predominant genus Prevo-tella, the family Lachnospiraceae, and the phylum Proteo-bacteria. Differences in primers used to generate amplicon libraries may be a major source of variations in microbiome profiling. Because different primers and regions of 16S rRNA genes are often used by different researchers, significant variations likely exist among studies. Quantitative interpre-tation for relative abundance of taxa, especially minor taxa, from prevalence of sequence reads and comparisons of re-sults from different studies should be done with caution.

• Keywords: 16S rRNA genes; microbiomes; primers; pyrose-quencing; sequencing facility