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Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization
Published on: April 14, 2016
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Modified PCR protocol to increase sensitivity for determination of bacterial community composition
Kayla M Williamson1, Brandie D Wagner1,2, Charles E Robertson3
1Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado School of Medicine, 13001 17th Place, Mail Stop B119, Aurora, CO, 80045, USA.
Microbiome
|April 14, 2021
Summary
A modified PCR protocol enhances bacterial detection in airway samples without altering community composition. This improved method is crucial for studying the variable airway microbiome in mechanically ventilated children.
Area of Science:
- Microbiology
- Genetics
- Bioinformatics
Background:
- Assessing bacterial communities in airway samples is challenging due to highly variable bacterial loads.
- Previous methods lacked the sensitivity to detect low bacterial concentrations.
- Mechanical ventilation in children often involves complex airway microbiome dynamics.
Purpose of the Study:
- To improve the sensitivity of sequence-based bacterial community determination.
- To evaluate two PCR protocols (standard and modified) for 16S rRNA gene amplification in airway samples.
- To ensure the modified protocol does not impact community composition or sequencing cluster formation.
Main Methods:
- Collected 99 tracheal aspirate (TA) samples from children requiring mechanical ventilation.
- Extracted DNA and quantified total bacterial load (TBL) using quantitative PCR (qPCR).
- Amplified 16S rRNA using both standard and modified PCR protocols.
Main Results:
- The modified PCR protocol successfully amplified bacterial DNA from 24 additional samples compared to the standard protocol.
- Amplification success correlated significantly with total bacterial load (p<0.001).
- Both protocols yielded similar community composition and low technical variability, with high agreement in diversity metrics.
Conclusions:
- The modified PCR protocol significantly increases sensitivity for bacterial community analysis in airway samples.
- This enhanced sensitivity is vital for studying the human airway microbiome, especially in low-biomass samples.
- The modified protocol offers a viable approach for more comprehensive microbiome analysis without compromising data integrity.
