A Metagenomic and Amplicon Sequencing Combined Approach Reveals the Best Primers to Study Marine Aerobic Anoxygenic Phototrophs

Abstract

Studies based on protein-coding genes are essential to describe the diversity within bacterial functional groups. In the case of 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 pufM gene amplification, design new ones, and evaluate their phylogenetic coverage. We then use samples from contrasting marine environments to evaluate their performance. By comparing the taxonomic composition of communities retrieved with metagenomics and with different amplicon approaches, we show that the commonly used PCR primers are biased towards the Gammaproteobacteria phylum and some Alphaproteobacteria clades. The metagenomic approach, as well as the use of other combinations of the existing and newly designed primers, show that these groups are in fact less abundant than previously observed, and that a great proportion of pufM sequences are affiliated to uncultured representatives, particularly 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.

Keywords: AAP bacteria; Amplicon sequencing; Metagenomics; Primer evaluation; pufM gene.

Fig. 1

Mismatches of primers pufMF [20], pufM_WAW, UniF, and UniR, [21], and pufMF_Y (this study) for the different AAP groups defined in this study. Dashed lines separate sequences with three or more mismatches

Fig. 2

A Sequence logo and phylogenetic coverage of primer pufMF [20]. The table shows the percentage of sequences with each nucleotide in the different positions of the primer, based on the alignment of our pufM database. Nucleotides representing positions with a high number of mismatches (≥ 19% of sequences) that could be associated with specific taxonomic groups (see color legend) are in bold and underlined. B Schematic representation of the pufM gene and the primers used in this study

Fig. 3

(A) Stations from the Malaspina Expedition used in this study (“MP” code). Samples from Blanes Bay Microbial Observatory (BBMO) are all from the same coastal site, yet collected at different times of the year. The code of Blanes samples indicates Blanes-year-month-day (e.g., BL110208 is from the 8th of February 2011). (B) pufM taxonomic composition of the BBMO left) and Malaspina (right) samples with the metagenomic approach. Below, the community composition at each station retrieved with the amplicon approach and with the different primers combinations: primers pufMF/pufM_WAW (C), pufMF_Y/pufM_WAW (D), and UniF/UniR (E). For each primer combination, and each sample, we have represented the relative abundances of the taxonomic groups retrieved with metagenomics vs. those retrieved with the amplicon approach. The dashed lines represent the 1:1 lines in which both the metagenomic and the amplicon approach would indicate the same taxonomic community composition