several show high similarities, suggesting they are the same species.
Coupled with the uncertain phenotypic/biochemical results shown
in Table 1, the species identification of these items cannot be
definitely resolved.
Fortunately, WGS yields more definitive results that allow differentiation and proper identification of these items. Table 2 provides digital DNA-DNA hybridization (dDDH) similarity values
obtained by using WGS (dDDH !70% indicates two genomes
belong to the same species; for more details on dDDH, see Subheading 4.3); the dDDH values are shown above the 100% selfcomparison diagonal. The table also includes M. tuberculosis variant
tuberculosis and M. tuberculosis variant bovis, as these two taxa
belong to the same species [17] and were included as an example
of two genomes that belong to the same species. The dDDH results
show that each of the remaining genomes are derived from taxa that
represent species distinct from each other, with similarity values all
well below the species-level threshold.
Thus, despite inconclusive results from phenotypic/biochemical testing and 16S rRNA similarities showing incorrect species
circumscriptions due to a limited single-gene resolving power,
WGS-based comparisons are able to provide accurate species circumscriptions and phylogenomic relationships based upon the
entirety of the organisms’ genomes.
3 Methods for Whole-Genome Sequencing
The overall process for whole-genome sequencing is shown in
Fig. 1. The quality and scientific utility of the resultant genomes
are inextricably linked to the methods used as each stage of the
sequencing process. This is often illustrated using the phrase “Garbage In, Garbage Out”. Using low-quality DNA as the input into
the process will typically yield lower-quality sequence. In order to
obtain the highest quality genomes possible, it is important that the
quality of the samples at each stage of the process is optimized.
Briefly, the organism to be sequenced is first acquired. Pure
cultures of type strains and known reference strains can be ordered
from culture collections across the globe for use as authenticated
and validated standards. Other common sources of mycobacterial
cultures include environmental isolates and of course clinical isolates, typically of the pathogenic species. Naturally, the nature of
such cultures as primary samples lends itself to contamination with
other organisms, either environmental or those that are a concomitant part of the human microbiome at the site of isolation. Unless
being studied within a metagenomics context, the mycobacterial
sample should be purified to isolation prior to sequencing.
Mycobacterial ID and Characterization Using WGS
403
Coupled with the uncertain phenotypic/biochemical results shown
in Table 1, the species identification of these items cannot be
definitely resolved.
Fortunately, WGS yields more definitive results that allow differentiation and proper identification of these items. Table 2 provides digital DNA-DNA hybridization (dDDH) similarity values
obtained by using WGS (dDDH !70% indicates two genomes
belong to the same species; for more details on dDDH, see Subheading 4.3); the dDDH values are shown above the 100% selfcomparison diagonal. The table also includes M. tuberculosis variant
tuberculosis and M. tuberculosis variant bovis, as these two taxa
belong to the same species [17] and were included as an example
of two genomes that belong to the same species. The dDDH results
show that each of the remaining genomes are derived from taxa that
represent species distinct from each other, with similarity values all
well below the species-level threshold.
Thus, despite inconclusive results from phenotypic/biochemical testing and 16S rRNA similarities showing incorrect species
circumscriptions due to a limited single-gene resolving power,
WGS-based comparisons are able to provide accurate species circumscriptions and phylogenomic relationships based upon the
entirety of the organisms’ genomes.
3 Methods for Whole-Genome Sequencing
The overall process for whole-genome sequencing is shown in
Fig. 1. The quality and scientific utility of the resultant genomes
are inextricably linked to the methods used as each stage of the
sequencing process. This is often illustrated using the phrase “Garbage In, Garbage Out”. Using low-quality DNA as the input into
the process will typically yield lower-quality sequence. In order to
obtain the highest quality genomes possible, it is important that the
quality of the samples at each stage of the process is optimized.
Briefly, the organism to be sequenced is first acquired. Pure
cultures of type strains and known reference strains can be ordered
from culture collections across the globe for use as authenticated
and validated standards. Other common sources of mycobacterial
cultures include environmental isolates and of course clinical isolates, typically of the pathogenic species. Naturally, the nature of
such cultures as primary samples lends itself to contamination with
other organisms, either environmental or those that are a concomitant part of the human microbiome at the site of isolation. Unless
being studied within a metagenomics context, the mycobacterial
sample should be purified to isolation prior to sequencing.
Mycobacterial ID and Characterization Using WGS
403