Inconsistent Denoising and Clustering Algorithms for Amplicon Sequence Data.

Natural microbial communities have been studied for decades using the 16S rRNA gene as a marker. In recent years, the application of second-generation sequencing technologies has revolutionized our understanding of the structure and function of microbial communities in complex environments. Using these highly parallel techniques, a detailed description of community characteristics are constructed, and even the rare biosphere can be detected. The new approaches carry numerous advantages and lack many features that skewed the results using traditional techniques, but we are still facing serious bias, and the lack of reliable comparability of produced results. Here, we contrasted publicly available amplicon sequence data analysis algorithms by using two different data sets, one with defined clone-based structure, and one with food spoilage community with well-studied communities. We aimed to assess which software and parameters produce results that resemble the benchmark community best, how large differences can be detected between methods, and whether these differences are statistically significant. The results suggest that commonly accepted denoising and clustering methods used in different combinations produce significantly different outcome: clustering method impacts greatly on the number of operational taxonomic units (OTUs) and denoising algorithm influences more on taxonomic affiliations. The magnitude of the OTU number difference was up to 40-fold and the disparity between results seemed highly dependent on the community structure and diversity. Statistically significant differences in taxonomies between methods were seen even at phylum level. However, the application of effective denoising method seemed to even out the differences produced by clustering.