Evaluating Quantitative Metagenomics for Environmental Monitoring of Antibiotic Resistance and Establishing Detection Limits
Benjamin C. Davis, Peter J. Vikesland, Amy Pruden
Abstract
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.