Benchmarking laboratory processes to characterise low-biomass respiratory microbiota

The low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA from reagents can alter the biological profile. The objective of this study was to benchmark the currently available protocols and laboratory workflows to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community composition, we amplified the 16S rRNA gene of respiratory samples using 16, 125 and 1000 pg of template DNA and 25, 30 and 35 PCR cycles. Libraries were purified by Agarose Gel Electrophoresis or AMPure XP and sequenced by V2 and V3 MiSeq reagent kits. The positive control was diluted in different solvents. The microbiota profiles of low biomass samples and DNA blanks were determined by Illumina MiSeq sequencing. PCR conditions had no significant influence on the microbial community composition of low biomass samples. Purification methods and MiSeq reagent kits had only a modest impact on microbiota profiles, while bacterial profiles of positive controls were significantly influenced by type of dilution solvent. Microbiota profiles of low biomass samples (16S rRNA gene concentration 0,1-1 pg/ul) can be accurately distinguished from DNA blanks. We conclude that microbiota profiling of low biomass samples is stable under several PCR conditions, different purification methods and different MiSeq reagent kits. We recommend to use amplification with 30 PCR cycles for low biomass samples. The amplicon pools can best be purified by two consecutive AMPure XP steps and sequenced by V3 MiSeq reagent kit. The benchmarked standardized workflow presented here ensures comparability of results within and between low biomass microbiome studies.