2025/03/18 by Benjamin C. Davis, Peter J. Vikesland, Amy Pruden · 1 voice
Biochemistry, Genetics and Molecular Biology · Engineering · Environmental Science · #Biosensors and Analytical Detection #Gut microbiota and health #Pharmaceutical and Antibiotic Environmental Impacts
paper · pdf · doi:10.1021/acs.est.4c08284
openalex publication_date 2025/03/18 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/26
High Resolution Image Download MS PowerPoint Slide Metagenomics holds promise as a comprehensive, nontargeted tool for environmental monitoring. However, one key limitation is that the quantitative capacity of metagenomics is not well-defined. Here, we demonstrated a quantitative metagenomic technique and benchmarked the approach for wastewater-based surveillance of antibiotic resistance genes. To assess the variability of low-abundance oligonucleotide detection across sample matrices, we spiked DNA reference standards (meta sequins) into replicate wastewater DNA extracts at logarithmically decreasing mass-to-mass percentages (m/m%). Meta sequin ladders exhibited strong linearity at input concentrations as low as 2 × 10 –3 m/m% ( R 2 > 0.95), with little to no reference length or GC bias. At a mean sequencing depth of 94 Gb, the limits of quantification (LoQ) and detection were calculated to be 1.3 × 10 3 and 1 gene copy per μL DNA extract, respectively. In wastewater influent, activated sludge, and secondary effluent samples, 27.3, 47.7, and 44.3% of detected genes were ≤LoQ, respectively. Volumetric gene concentrations and log removal values were statistically equivalent between quantitative metagenomics and ddPCR for 16S rRNA, intI 1, sul 1, CTX-M-1, and van A. The quantitative metagenomics benchmark here is a key step toward establishing metagenomics for high-throughput, nontargeted, and quantitative environmental monitoring.