Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure

Abstract

PCR-based surveys of microbial communities commonly use regions of the small subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using OTU- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of ca. 100 base pair (bp), 400bp, and 1000bp from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates, and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias, and mis-priming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.