Low-biomass microbiome kit evaluation. Low-biomass microbiome kit evaluation. (a) Experimental design of low-biomass kit evaluating negative and positive controls (5, 50, 500, and 5,000 bacterial cells) across three solid-phase and two magnetic-bead-based DNA extraction methods. LT, low throughput; HT, high throughput. (b) Libraries of 16S rRNA amplicons were sequenced, and the limit of detection of B. subtilis from DNA extraction was determined by comparing the composition of the expected target in known inputs of cells with that in negative controls by a nonparametric Kruskal-Wallis test (Benjamini-Hochberg FDR of 0.05). (c) The limit of detection of the assay and approximate background noise were determined by calculating the K1/2 value by using the allosteric sigmoidal equation on the Bacillus composition. (d) The median read counts (interquartile range) were compared against blanks by using a nonparametric Kruskal-Wallis test (Benjamini-Hochberg FDR of 0.05) to distinguish signal from noise. (e) Read counts were plotted against the expected composition of the target and fitted with an allosteric sigmoidal equation to describe the number of reads where 50% of the read composition was the expected target. This was performed for DNA extraction positive controls from the Mo Bio PowerMag kit, which performed the best, with a limit of detection of 50 cells. Jeremiah J. Minich et al. mSystems 2018; doi:10.1128/mSystems.00218-17