Evaluating Quantitative Metagenomics for Environmental Monitoring of Antibiotic Resistance and Establishing Detection...

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% (R² > 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 × 10³ 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, intI1, sul1, CTX-M-1, and vanA. The quantitative metagenomics benchmark here is a key step toward establishing metagenomics for high-throughput, nontargeted, and quantitative environmental monitoring.