DNA template dilution impacts amplicon sequencing-based estimates of soil fungal diversity

Next generation sequencing of taxonomically relevant marker genes has enabled researchers to sample the richness, diversity, and composition of environmental microbiomes at previously unattainable depths. However, molecular methods may have unintended downstream consequences and the inadvertent undersampling of microbial communities may be a significant pitfall in microbiome profiling. One such procedure, dilution of the DNA template prior to polymerase chain reaction (PCR), may improve marker gene amplification by reducing chimeric read formation and decreasing PCR inhibitor concentrations. However, dilution unavoidably reduces target DNA template number per sample. We evaluated the effects of pre-PCR DNA template dilution on estimates of soil fungal microbiome diversity, composition, and species abundance distributions across a collection of 144 agricultural soil samples. Fungal DNA templates were serially diluted at 0-, 10-, 100-, and 1,000-fold and sequence data of diluted templates were compared with those of an identical set of undiluted templates. For three prairie soil samples, in addition to evaluating variation among replicates of individual samples, we serially diluted fungal DNA extracts from soil samples in triplicate and sequenced undiluted and diluted samples. DNA template dilution significantly reduced estimates of fungal richness and diversity, as compared with undiluted samples. Dilution of DNA template also resulted in reduced relative abundances of rare operational taxonomic units (OTUs) and increased relative abundances of common OTUs. Collectively, changes in OTU abundance distributions following dilution produced substantial shifts in overall fungal community composition. Our results highlight risks associated with sample dilution and point to the potential utility of quantifying pre-PCR template concentration in the estimation of microbiomes. We urge researchers to thoroughly document methods and to reconsider routine dilution of pre-PCR DNA templates particularly for low abundance microbiome samples. As efforts to profile environmental microbiomes using molecular sequencing approaches accelerate, developing an adequate understanding of potential methodological bottlenecks will increase our ability to accurately characterize and compare datasets.