Table 1.

Metagenomic approaches for pathogen detection and their findings and applications

	Method	Applications	Recent examples	Advantages	Limitations
Deep amplicon sequencing	• rRNA	• Prokaryotic and eukaryotic identification*	• Characterization of the healthy human gut microbiome (HMP) [28]	• Potentially higher sensitivity	• Targeted gene may not be truly universal
		• Determination of taxonomic relationships	• Ancient gut microbiomes found to be more similar to modern rural than modern cosmopolitan microbiomes [29]	• Less expensive as fewer reads are required for taxonomic classification	• Primer bias may alter population structure
	• rpoB	• Archaeal and bacterial identification*	• Used to divide the species Gardnerella vaginalis into subgroups [30]	• rpoB and cpn-60 offer enhanced taxonomic resolution compared to rRNA [31,32]	• Possibility of variable gene copy numbers amongst targeted species
	• cpn-60	• Determination of taxonomic relationships
	• Viral RNA polymerase (RdRP)	• Novel virus discovery	• Identified novel families of picornaviruses off the coast of British Columbia [33]
Metagenomics	• Shotgun sequencing	• Functional and taxonomic characterization	• Detection of African swine fever virus-like sequences representing new members of the family Asfariviridae [9]	• Recovery of sequences from all microorganisms	• Broad specificity might decrease sensitivity
			• Detection of unexpected microbes from stool samples [12]	• No a priori knowledge of microorganisms required	• Library preparation is relatively labor intensive
	• Subtraction	• Functional and taxonomic characterization	• Identified divergent regions in non-coding RNAs in Listeria monocytogenes[34]	• Random primers reduce potential for bias	• Bioinformatics analysis is more challenging
			• Association of Fusobacterium nucleatum with colorectal carcinoma [35]		• Relatively expensive as more reads are required than for DAS
	• Virus concentration	• Novel virus discovery	• Detection of the novel H1N1 influenza from nasopharyngeal swabs [13]		• Approximately 50% of sequences generally have no significant homology to known proteins in databases (dark matter) [36]
			• Detection of a novel rhabdovirus from serum [37]
	• Hybridization capture	• Investigation of sequences with very low copy number	• Metagenomic analysis of tuberculosis from a mummy [38]		• Increased granularity in population structure determination [39]
			• Investigation of Yersinia pestis from ancient teeth [40]

*Specific primers need to be made to discriminate between each group. RdRP, RNA-dependent RNA polymerase.