Examining changes in bacterial abundance in complex communities using next-generation sequencing is enhanced with...

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Microbial community composition is frequently studied using high-throughput sequencing (HTS).
• Accurate quantification of specific taxa within these communities can be challenging.
• Dominant taxa may influence the interpretation of HTS data.

Purpose of the Study:

• To evaluate the accuracy of HTS for determining relative microbial abundance.
• To compare HTS data with quantitative PCR (qPCR) for absolute abundance measurements.
• To assess the impact of combining HTS and qPCR in analyzing microbial communities.

Main Methods:

• Analysis of bar-coded Illumina sequencing data from cystic fibrosis patient sputum.
• Comparison of relative abundance from HTS with absolute abundance from qPCR.
• Investigated specific taxa: *Pseudomonas aeruginosa* and *Burkholderia multivorans*.

Main Results:

• No significant correlation was found between the relative abundance of *P. aeruginosa* or *B. multivorans* derived from HTS and their absolute abundance measured by qPCR.
• Conclusions drawn from HTS data alone differed from those obtained by combining HTS and qPCR.
• HTS data alone may not accurately reflect the true abundance of specific organisms in complex microbial communities.

Conclusions:

• Quantitative PCR (qPCR) is essential when using high-throughput sequencing (HTS) for microbial community analysis.
• Combining HTS and qPCR provides a more accurate characterization of microbial communities, particularly those with dominant taxa.
• This integrated approach improves the reliability of findings in microbiome research.