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Metagenomic Quantification of Genes with Internal Standards
Emily Crossette1, Jordan Gumm2, Kathryn Langenfeld1
1Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan, USA.
Mbio
|February 3, 2021
Summary
A new quantitative metagenomic method accurately measures absolute gene concentrations in environmental samples, offering a high-throughput alternative to quantitative PCR (qPCR) for tracking antimicrobial resistance genes (ARGs). This approach detects thousands of genes, providing valuable insights into microbial communities.
Area of Science:
- Environmental microbiology
- Molecular biology
- Genomics
Background:
- Quantitative PCR (qPCR) and metagenomics are key techniques for studying gene dynamics and tracking antimicrobial resistance genes (ARGs).
- A need exists for tools capable of quantifying thousands of genes per sample mass or volume.
Purpose of the Study:
- To compare a quantitative metagenomic approach with traditional qPCR for ARG quantification in dairy manure.
- To demonstrate high-throughput absolute gene quantification of all known ARG sequences in environmental samples.
Main Methods:
- Developed an assembly-independent, spike-in facilitated metagenomic quantification approach.
- Extracted DNA from dairy manure, spiked with marine bacterium DNA, and sequenced using Illumina HiSeq4000.
- Compared gene copy concentrations of five ARGs using metagenomics, qPCR, and a hybrid approach.
Main Results:
- The metagenomic method delivered absolute gene concentrations comparable to qPCR.
- Metagenomics avoided primer biases inherent to qPCR and detected orders of magnitude more genes.
- Observed consistent total abundance of tetracycline resistance genes across manure treatment stages, with varying dominant gene families.
Conclusions:
- The quantitative metagenomic approach enables simultaneous, high-throughput absolute quantification of thousands of genes.
- This method offers a powerful alternative to qPCR for comprehensive ARG profiling in environmental samples.
