Table 2.

Metagenomic sequencing vs. other techniques used to study qualitatively and quantitatively antibiotic resistance.

	Whole-genome sequencing	Meta-proteomics	Whole-metagenome sequencing	Quantitative PCR
Definition	The sequencing of an entire genome of a single organism is obtained from a culture to identify species/ strains, genes, and mutations associated with AMR.	Identification and quantification of proteins conferring AMR and Multi-drug resistance (MDR) to localized MGEs from microbial communities.	A culture-independent approach is used for the identification of all ARGs/ mutations in all organisms present in complex microbial communities.	Method for the detection, quantification, and typing of specific microbial species/ strains or ARGs/ markers.
Technique	DNA is extracted from bacterial culture and sequenced to generate FastQ reads.	The generation of peptides and high-resolution mass spectra analysis to identify and quantify proteins.	DNA is extracted directly from a sample and sequenced to generate reads representing the entire microbial community.	An enzymatic reaction in combination with fluorophores (SYBR Green and TaqMan) is used to amplify a specific target of interest and quantify gene expression levels.
Advantages	+ Reproducible High resolution Standardization of analysis methods Rapid turn-around time High specificity Read lengths of 1 – 50Kb	Accurate prediction Large-scale protein identification Rapid turn-around time	+ High resolution Accurate prediction	Fast High-throughput detection and quantification of target DNA sequences avoid cross-contamination Sensitivity Specificity
Limitations	- Requires cultured organism Computationally demanding Cost	- False positives from large datasets False discovery rates in large datasets Limited reproducibility	- Difficult to differentiate between host and pathogen Expensive Computationally demanding	- Incapable of distinguishing between live and dead cells
Outcome	Microbial typing and tracing Ability to predict AMR from only genomic data Discovery of novel resistance genes or mutations	Host-pathogen interaction Virulence factors Antibiotic resistance mechanisms Identification of functions involved in biological pathways	Species-or strain level identification AMR Virulence potential Detect Mobile genetic elements Discovery of viruses	Determine the presence of specific genes and alleles AMR profiling Toxin production Typing of strains and isolates