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O’Connell et al. Microbiome Res Rep 2023;2:21 https://dx.doi.org/10.20517/mrr.2023.17 Page 19 of 21

single phage presenting the subcluster G5 was recommended for inclusion in subcluster G3 as it met the
VIRIDIC (≥ 70%), Gegenees (~ 50%) and VICTOR (monophyletic branch) parameters for inclusion in
subcluster G3. Overall, it appears as though the criteria for genus inclusion are adequate to support the
creation (or abolition) of subclusters, thereby formalising this classification.

While the 721 MP selected for this study are a small cohort of the more than 12,000 isolated MP, they
represent approximately one-third of sequenced MP, which is a sizeable sample size. Although the genus-
subcluster link is not infallible - and the limitations of DNA- and proteome-based comparisons along with
genetic mosaicism which has been briefly discussed should not be completely disregarded, novel genus
assignment appears to be a reliable indicator of subcluster creation. The original 83.3% of the dataset that
supported the hypothesis increased to 97.6% when the 20 novel genera and 13 novel subclusters identified in
this study were considered. Overall, these results highlight the necessity to frequently revise taxonomic
classifications (potentially as a routine feature of novel phage genome characterisation) and ensure the
fidelity of cluster and subcluster assignments as phage taxonomy evolves and more of the viral biosphere is
characterised. By recognising and maintaining the genus-subcluster relationship between the taxonomic
and clustering classification systems as much as possible, it will ensure that the diversity of MP is accurately
reflected in both systems as more MP are sequenced and novel MP are isolated. Robust and linked
classification systems could then aid rapid phage selection for research, therapeutic and diagnostic purposes
as closely related phage will be easily defined within a cluster.

DECLARATIONS
Authors’ contributions
Made substantial contributions to the conception and design of the study and performed data analysis and
interpretation: O’Connell LM, Buttimer C
Performed data acquisition, as well as provided administrative, technical, and material support: Bottacini F
Provided research guidance and revisions of draft manuscripts prior to submission: Coffey A, O’Mahony
JM

Availability of data and materials
The original taxonomy and cluster/subcluster assignments of the 721 mycobacteriophages included in the
dataset and the changes proposed in this study are available in Supplementary Table 1.

Financial support and sponsorship
This study was supported by the Risam PhD Scholarship awarded by Munster Technological University.

Conflicts of interest
All authors declared that there are no conflicts of interest.

Ethical approval and consent to participate
Not applicable.

Consent for publication
Not applicable.

Copyright
© The Author(s) 2023.