To study the effect of PCR conditions on the microbial community composition, we amplified the 16S rRNA gene of respiratory samples using various 16S rRNA gene bacterial load input and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 and V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents.
PCR conditions had no significant influence on the microbial community composition of low biomass samples. Purification methods gave nearly similar microbiota profiles (paired Bray-Curtis dissimilarity median: 0.03). MiSeq reagent kits V2 vs V3 showed a high concordance (paired Bray-Curtis dissimilarity median: 0.05 vs unpaired Bray-Curtis dissimilarity median: 0.8). Profiles of positive controls were significantly influenced by type of dilution solvent, the theoretical Zymo mock bacterial composition was most closely resembled by Zymo mock diluted in elution buffer (difference: 21,6% elution buffer, 79,6% DNA/RNA shield and 29,2% Milli-Q). DNA blanks represent a distinct cluster compared to microbiota profiles of low biomass samples.
We recommend to use amplification with 30 PCR cycles. The amplicon pools can best be purified by two consecutive AMPure XP steps and sequenced by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.