Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes

Abstract

Operational taxonomic units (OTUs) are conventionally defined at a phylogenetic distance (0.03—species, 0.05—genus, 0.10—family) based on full-length 16S rRNA gene sequences. However, partial sequences (700 bp or shorter) have been used in most studies. This discord may affect analysis of diversity and species richness because sequence divergence is not distributed evenly along the 16S rRNA gene. In this study, we compared a set each of bacterial and archaeal 16S rRNA gene sequences of nearly full length with multiple sets of different partial 16S rRNA gene sequences derived therefrom (approximately 440–700 bp), at conventional and alternative distance levels. Our objective was to identify partial sequence region(s) and distance level(s) that allow more accurate phylogenetic analysis of partial 16S rRNA genes. Our results showed that no partial sequence region could estimate OTU richness or define OTUs as reliably as nearly full-length genes. However, the V1–V4 regions can provide more accurate estimates than others. For analysis of archaea, we recommend the V1–V3 and the V4–V7 regions and clustering of species-level OTUs at 0.03 and 0.02 distances, respectively. For analysis of bacteria, the V1–V3 and the V1–V4 regions should be targeted, with species-level OTUs being clustered at 0.04 distance in both cases.