implementation employed segmentation of the query genome
sequence into fragments of 1020 bp; these fragments are then
used to query the reference genome [90]. The threshold for species
circumscription by ANI is approximately !95–96% [93–95]. Various different “flavors” of ANI have been described, including
OrthoANI, which addresses the nonreciprocity of ANI calculations
with a modified algorithm that provides orthologous ANI values,
[96] and FastANI, which alleviates the bottleneck caused by computationally expensive alignment using Mashmap to enable
sequence mapping in an alignment-free manner [95].
Digital DNA-DNA hybridization (dDDH) is an in silico adaptation of DDH that allows genome-to-genome distances (GGDs)
to be calculated in a pairwise manner from whole-genome DNA
sequences [87, 97]. dDDH is algorithmically calibrated to the same
scale as DDH, with 70% genome similarity representing membership in the same species. In addition to this species-level threshold,
dDDH provides a subspecies-level threshold of 79–80%
[98]. dDDH is publicly available through a service called the
Genome-to-Genome Distance Calculator (GGDC).
By using either ANI or dDDH to compare two genomes to
each other, one can determine whether those two genomes belong
to the same species or not. However, unless used properly, this
method does not indicate which species that is. The choice of
reference genome to be compared against is key. In order to properly identify a sample to the species or subspecies level, the reference
genome used must be from the type strain of the species or subspecies. For the researcher’s convenience, a list of type strain genomes
is provided in Table 3.
The increasing ease with which high-quality genomes can be
produced coupled with a steadily decreasing cost has led to an
explosion in the number of genomes. This has led to numerous
predictions that WGS-based analysis will soon replace wet-lab
DDH and numerous recommendations for this to be enacted
[16, 86, 87, 90, 92, 94, 97, 99–102]. However, even without a
formal designation as the new gold standard, genomic analysis
specifically in the form of ANI and dDDH appears to have begun
cementing its place as a new de facto gold standard. An examination
of the papers published in the International Journal of Systematic
and Evolutionary Microbiology in which novel taxa are named or
existing taxa are reclassified shows that the number of papers using
such digital techniques continues to increase essentially every
month. Indeed, at the time of this writing, the most recent issue
of IJSEM available (January 2020) includes 91 articles identifying
novel bacterial species or reclassifying bacterial species; of these,
78 (85.7%) base their analysis in part on results from ANI and/or
dDDH. A similar percentage (38/42 articles ¼ 90.5%) is observed
in the December 2019 issue. This reflects not only the increasing
Mycobacterial ID and Characterization Using WGS
423
sequence into fragments of 1020 bp; these fragments are then
used to query the reference genome [90]. The threshold for species
circumscription by ANI is approximately !95–96% [93–95]. Various different “flavors” of ANI have been described, including
OrthoANI, which addresses the nonreciprocity of ANI calculations
with a modified algorithm that provides orthologous ANI values,
[96] and FastANI, which alleviates the bottleneck caused by computationally expensive alignment using Mashmap to enable
sequence mapping in an alignment-free manner [95].
Digital DNA-DNA hybridization (dDDH) is an in silico adaptation of DDH that allows genome-to-genome distances (GGDs)
to be calculated in a pairwise manner from whole-genome DNA
sequences [87, 97]. dDDH is algorithmically calibrated to the same
scale as DDH, with 70% genome similarity representing membership in the same species. In addition to this species-level threshold,
dDDH provides a subspecies-level threshold of 79–80%
[98]. dDDH is publicly available through a service called the
Genome-to-Genome Distance Calculator (GGDC).
By using either ANI or dDDH to compare two genomes to
each other, one can determine whether those two genomes belong
to the same species or not. However, unless used properly, this
method does not indicate which species that is. The choice of
reference genome to be compared against is key. In order to properly identify a sample to the species or subspecies level, the reference
genome used must be from the type strain of the species or subspecies. For the researcher’s convenience, a list of type strain genomes
is provided in Table 3.
The increasing ease with which high-quality genomes can be
produced coupled with a steadily decreasing cost has led to an
explosion in the number of genomes. This has led to numerous
predictions that WGS-based analysis will soon replace wet-lab
DDH and numerous recommendations for this to be enacted
[16, 86, 87, 90, 92, 94, 97, 99–102]. However, even without a
formal designation as the new gold standard, genomic analysis
specifically in the form of ANI and dDDH appears to have begun
cementing its place as a new de facto gold standard. An examination
of the papers published in the International Journal of Systematic
and Evolutionary Microbiology in which novel taxa are named or
existing taxa are reclassified shows that the number of papers using
such digital techniques continues to increase essentially every
month. Indeed, at the time of this writing, the most recent issue
of IJSEM available (January 2020) includes 91 articles identifying
novel bacterial species or reclassifying bacterial species; of these,
78 (85.7%) base their analysis in part on results from ANI and/or
dDDH. A similar percentage (38/42 articles ¼ 90.5%) is observed
in the December 2019 issue. This reflects not only the increasing
Mycobacterial ID and Characterization Using WGS
423