Critical Relevance of Stochastic Effects on Low-Bacterial-Biomass 16S rRNA Gene Analysis.

ABSTRACT: The bacterial microbiome of human body sites, previously considered sterile, remains highly controversial because it can be challenging to isolate signal from noise when low-biomass samples are being analyzed. We tested the hypothesis that stochastic sequencing noise, separable from reagent contamination, is generated during sequencing on the Illumina MiSeq platform when DNA input is below a critical threshold. We first purified DNA from serial dilutions of Pseudomonas aeruginosa and from negative controls using three DNA purification kits, quantified input using droplet digital PCR, and then sequenced the 16S rRNA gene in four technical replicates. This process identified reproducible contaminant signal that was separable from an irreproducible stochastic noise, which occurred as b

SUBMITTER: Erb-Downward JR 

PROVIDER: S-EPMC7373192 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature