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

MBio
John R Erb-DownwardSara D Adar

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 bacterial biomass of samples decreased. This approach was then applied to authentic respiratory samples from healthy individuals (n = 22) that ranged from high to ultralow bacterial biomass. Using oral rinse, bronchoalveolar lavage (BAL) fluid, and exhaled breath condensate (EBC) samples and matched controls, we were able to demonstrate (i) that stochastic noise dominates sequencing in real-world low-bacterial-biomass samples that contain fewer than 1...Continue Reading

Associated Clinical Trials

References

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Citations

Jan 14, 2021·Microbiome·Rachel B Silverstein, Indira U Mysorekar
Nov 13, 2020·Cancer Discovery·Jun-Chieh J TsayLeopoldo N Segal
May 12, 2021·Nature Microbiology·Katherine M KennedyThorsten Braun
Jan 16, 2021·The European Respiratory Journal·Imran SulaimanLeopoldo N Segal
Aug 3, 2021·The Journal of Clinical Investigation·Samantha A WhitesideRonald G Collman
Aug 8, 2021·Microorganisms·Aurélien AlafaciJulien Verdon
Jun 24, 2021·American Journal of Physiology. Lung Cellular and Molecular Physiology·Jay H LipinskiDavid N O'Dwyer

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Datasets Mentioned

BETA
PRJNA549253
PRJNA552077

Methods Mentioned

BETA
amplicon sequencing
bronchoalveolar lavage
PCA
PCR

Software Mentioned

R
ComplexHeatmap
RColorBrewer
tidyr
ggplot2
dplyr
mvabund
Prism
vegan
GraphPad

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