A metagenomics and amplicon sequencing combined approach reveals the best primers to study marine aerobic anoxygenic phototrophs

Abstract

Abstract Studies based on protein-coding genes are essential to describe the diversity within bacterial functional groups. In the case of the aerobic anoxygenic phototrophic (AAP) bacteria, the pufM gene has been established as the genetic marker for this particular functional group, although available primers are known to have amplification biases. We review here the existing primers for the pufM gene amplification, design new ones, and evaluate their phylogenetic coverage. We then use samples from different marine environments to evaluate the performance of different primer combinations. By comparing the taxonomic composition of communities retrieved with PCR-free metagenomics and with the different amplicon approaches, we show that the commonly used primers are biased towards the Gammaproteobacteria phylum and some Alphaproteobacteria clades. The metagenomic approach as well as other combinations of the existing and newly designed primers show that these groups are in fact a minority and a great proportion of pufM sequences are affiliated to uncultured representatives, especially in the open ocean. Altogether, the framework developed here becomes a better alternative for future studies based on the pufM gene, and additionally serves as a reference for primer evaluation of other functional genes.