Modified PCR protocol to increase sensitivity for determination of bacterial community composition.

BACKGROUND: The objective of this project was to increase the sensitivity of sequence-based bacterial community determination without impacting community composition or interfering with cluster formation during sequencing. Two PCR protocols (standard and modified) were examined in airway samples where we observed a large range in bacterial load (3.1-6.2 log10 16S rRNA gene copies/reaction). Tracheal aspirate (TA) samples (n = 99) were collected from sixteen children requiring mechanical ventilation at a single center. DNA was extracted, and total bacterial load (TBL) was assessed using qPCR. Amplification of 16S rRNA was attempted with both protocols in all samples. RESULTS: PCR product was observed using both protocols in 52 samples and in 24 additional samples only with the modified protocol. TBL, diversity metrics, and prominent taxa were compared for samples in three groups based on success of the two protocols (successful with both, success with modified only, unsuccessful for both). TBL differed significantly across the three groups (p<0.001). Specifically, the modified protocol allowed amplification from samples with intermediate TBL. Shannon diversity was similar between the two protocols, and Morisita-Horn beta diversity index showed high agreement between the two protocols within samples (median value 0.9997, range 0.9947 to 1). We show that both protocols identify similar communities, and the technical variability of both protocols was very low. The use of limited PCR cycles was a key feature to limit impact of background by exclusion of 24% of samples with no evidence of bacterial DNA present in the sample. CONCLUSION: The modified amplification protocol represents a viable approach that increased sensitivity of bacterial community analysis, which is important for study of the human airway microbiome where bacterial load is highly variable. Video abstract.