Mock community as an in situ positive control for amplicon sequencing of microbiotas from the same ecosystem

Abstract

Metataxonomy has become the standard for characterizing the diversity and composition of microbial communities associated with multicellular organisms and their environment. Currently available protocols for metataxonomy assume a uniform DNA extraction, amplification and sequencing efficiency for all sample types and taxa. It has been suggested that the addition of a mock community (MC) to biological samples before the DNA extraction step could aid identification of technical biases during processing and support direct comparisons of microbiota composition, but the impact of MC on diversity estimates of samples is unknown. Here, large and small aliquots of pulverized bovine fecal samples were extracted with no, low or high doses of MC, characterized using standard Illumina technology for metataxonomics, and analysed with custom bioinformatic pipelines. We demonstrated that sample diversity estimates were distorted only if MC dose was high compared to sample mass (i.e. when MC > 10% of sample reads). We also showed that MC was an informative in situ positive control, permitting an estimation of the sample 16S copy number, and detecting sample outliers. We tested this approach on a range of sample types from a terrestrial ecosystem, including rhizosphere soil, whole invertebrates, and wild vertebrate fecal samples, and discuss possible clinical applications.