Fig. 2

16S-RNA-seq was not able to differentiate active vs. whole microbiome in spiked realistic communities. Our second evaluation of 16S-RNA-seq used four environmental microbial community types (high and low biomass and high and low expected activity) spiked with varying levels of cultured/heat-killed E. coli. a Relative abundances of 15 taxa detected with the highest mean abundance across all samples with clear differences between sample types. Four biological replicates were taken for computer screens and mice and three biological replicates for saliva and soil. b Bray–Curtis dissimilarity within and between communities from DNA and RNA libraries indicated no significant differences between the RNA (cDNA) and DNA pairs (PERMANOVA R²: 2.3%, FDR q: 0.254). The RNA (cDNA) libraries showed the highest inter-replicate dissimilarity, followed by the DNA in most cases. Columns labeled with the “sample_DNA” (e.g., Screen_DNA) show dissimilarities within the indicated DNA libraries. Those annotated “type_RNA” (e.g., Screen_RNA) show calculations within the RNA libraries, and “type_between” (e.g., Screen_between) represents distances between paired samples in DNA vs. RNA libraries. c After constructing an ordination based on each sample’s pairwise Bray–Curtis dissimilarity, the dissimilarities were largely explained by sample types. With the screens and mice ordinating closest together, as expected. Lines connect identical samples in DNA and RNA libraries