| 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] |  |  |