Measuring both microbial load and diversity with a single amplicon sequencing library

The microbial population size, or load, in a host is a fundamental metric of colonization by commensals or infection by pathogens. Sequence-based measurement of DNA amount contributed by host and microbes in a sample provides a simple way of measuring microbial load, and it also has the ability to estimate microbiome diversity. Unfortunately, it is also very costly, especially when host DNA greatly outweighs microbial DNA. We introduce a robust two-step PCR strategy to quantify microbial density of the host-associated microbiome and describe its composition in a single, cost-effective amplicon library. We demonstrate the accuracy and flexibility of this method across multiple amplicons and hosts, and further present a simple technique that can be used, prior to sequencing, to optimize the host representation in a batch of libraries without a loss of information.