Current Applications of Absolute Bacterial Quantification in Microbiome Studies and Decision-Making Regarding Different Biological Questions.

High throughput sequencing has emerged as one of the most important techniques for characterizing microbial dynamics and revealing bacteria and host interactions. However, data interpretation using this technique is mainly based on relative abundance and ignores total bacteria load. In certain cases, absolute abundance is more important than compositional relative data, and interpretation of microbiota data based solely on relative abundance can be misleading. The available approaches for absolute quantification are highly diverse and challenging, especially for quantification in differing biological situations, such as distinguishing between live and dead cells, quantification of specific taxa, enumeration of low biomass samples, large sample size feasibility, and the detection of various other cellular features. In this review, we first illustrate the importance of integrating absolute abundance into microbiome data interpretation. Second, we briefly discuss the most widely used cell-based and molecular-based bacterial load quantification methods, including fluorescence spectroscopy, flow cytometry, 16S qPCR, 16S qRT-PCR, ddPCR, and reference spike-in. Last, we present a specific decision-making scheme for absolute quantification methods based on different biological questions and some of the latest quantitative methods and procedure modifications.