Alignment-free phylogenetic inference can determine subcluster assignments of phages only when using entire genome sequences. As predicted, a feature frequency profile (FFP) can identify subclusters when given sufficient nucleotide sequences for the analysis, such as entire phage genomes (A); however, the TMP gene sequence is too short for the feature frequency profile to identify relationships (B). The geneological sorting index (gsi) for clades indicates subclusters are identified well in the whole genome analysis and poorly or not at all in the TMP analysis (C). The mycobacteriophage genomes used were identical to the 79 genomes used throughout this study, which represent 30 mycobacteriophage subclusters. Feature frequency profiles  were used to infer phylogenetic relationships [19–21] using Bacillus cereus PBC1 phage as outgroup. The neighbor-joining method was used to infer a phylogeny which was bootstrapped 10,000 times to assess nodal support. A 50% majority-rule consensus tree was obtained using Paup* 4.0  and annotated in FigTree 1.3.1 (http:// tree.bio.ed.ac.uk/software/figtree).