Evaluation of a new primer combination to minimize chloroplast plastid contamination in 16S rDNA metabarcoding analyses of alga-associated bacterial communities

The diversity of bacterial communities associated with plant and algal hosts is generally described by 16S rDNA metabarcoding approaches targeting the 16S rDNA via primer sets universal to Bacteria. The limit of these primers is that the resulting datasets frequently contains many chloroplast or plastidial sequences. This is because chloroplasts plastid genomes, deriving from endosymbiosis events, still encode 16S rDNA closely related to their cyanobacterial ancestors. To avoid the amplification of chloroplasts plastid-related sequences we performed in silico tests of the taxonomic coverage of four primer combinations targeting the variable V3-V4 region of the 16S rDNA. They included a forward primer universal to Bacteria and four reverse primers designed to avoid chloroplastsplastids. The best primer combination (NOCHL) was then compared to the universal primer set in vitro using a synthetic community of 32 bacterial strains and samples from 3 brown macroalgale species. Overall, the relative abundance of chloroplast plastid sequences was reduced by 99-100% with the NOCHL primers while the observed community structure was maintained. The phyla Planctomycetes, Verrucomicrobia, and Cyanobacteria were underrepresented compared to the universal primer. Therefore, our NOCHL primer set is efficient to avoid amplification of chloroplast plastid 16S rDNAs and may need to be combined with general or taxon-specific primers for a complete description of the alga-associated bacterial diversity.