BENEFITS AND CHALLENGES OF QUANTIFYING MICROBIAL GENE COPIES IN THE OCEAN IN THE ERA OF 'OMICS

Accurately quantifying variability in the community composition and function of marine microbial populations remains a major challenge. Metagenomics and -transcriptomics provide detailed information about the composition and functional potential of marine microbial communities without PCR amplification bias, whereas high-throughput amplicon sequencing targeting ribosomal or functional genes provides detailed information about community composition and activity, and is a standard and widely used tool in comparative analyses of marine microbial communities. Yet in all of the high-throughput sequencing-based methods, underlying changes in actual abundances of individual microbial target groups in mixed microbial communities are unknown, which can easily lead to interpretation biases. Many key processes in marine biogeochemical cycles are conducted by organisms found at relatively low abundances in the context of total cell abundances, and are often missed in a bulk ‘omics sequencing. In addition, the associated costs constrain ‘omics analyses to relatively few samples per study. Although not without its own biases, Quantitative Polymerase Chain Reaction (qPCR) arguably remains the most widely available molecular approach for specifically detecting and quantifying abundances of microbial taxon-specific gene copies, thereby providing a proxy for cell abundances. Through the activity under the Ocean Carbon Biogeochemistry Workgroup on nitrogen fixation, we have developed recommendations on the procedures for conducting qPCR with seawater DNA and RNA samples, covering steps from sample collection to analytical procedures and calculations of copy numbers. We also highlight known biases and weaknesses of the method that are important to consider in interpretation and extrapolation of environmental qPCR data on taxon-specific distributions and activity.