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Related Experiment Video

Updated: Jan 4, 2026

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Published on: June 23, 2019

Microbiota analysis optimization for human bronchoalveolar lavage fluid.

Pierre H H Schneeberger^1,2, Janice Prescod^3,4, Liran Levy^3,4

¹Departments of Medicine and Laboratory Medicine & Pathobiology, University of Toronto, Toronto, M5G 1L7, Canada. pierre.schneeberger@gmail.com.

|October 31, 2019

Summary

Investigating lung microbiota with 16S sequencing requires careful consideration of sample density. Contamination in bronchoalveolar lavage fluid (BALF) can skew results, but pre- and post-sequencing steps can improve accuracy for samples above 8E+04 16S copies/ml.

Keywords:

Lung microbiota Sequencing accuracy Sequencing precision, bronchoalveolar lavage

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Last Updated: Jan 4, 2026

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Published on: June 23, 2019

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

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Published on: May 4, 2017

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

Published on: March 24, 2014

Area of Science:

Microbiology
Genomics
Bioinformatics

Background:

Culture-independent sequencing assays allow comprehensive lung microbiota characterization.
Bronchoalveolar lavage fluid (BALF) is used to study lower lung microbiota but often has low bacterial density, risking contamination.
Contaminating DNA can lead to false-positive signals in BALF 16S sequencing data.

Purpose of the Study:

To assess contamination in BALF samples across various densities.
To identify contaminant features for improved data interpretation.
To optimize 16S sequencing analysis of BALF samples.

Main Methods:

Mock communities with densities from 8E+02 to 8E+09 16S copies/ml were sequenced.
Taxonomic accuracy, precision, and contaminant proportions were evaluated.
Pre- and post-sequencing steps were tested for contaminant removal.

Main Results:

Sequencing accuracy and precision decreased below 8E+05 16S copies/ml.
Contaminants were often inversely correlated with sample density or absent in replicates.
Pre- and post-sequencing steps improved accuracy for samples >8E+04 16S copies/ml.
Low-density BALF samples (<8E+03 16S copies/ml) showed unresolvable contamination.

Conclusions:

BALF 16S sequencing data interpretation must consider sample density.
Accuracy and precision can be enhanced for samples >8E+04 16S copies/ml using specific steps.
Contaminant OTUs observed in mock communities were also present in low-density clinical BALF samples.