*3.1. Distribution of HPV 16 Sub-Lineages in Anal Cancers*

Of the 119 cancer cases with sufficient read depth (>100× median depth and >80% genome), the HPV 16 sub-lineage A1 was identified in 91 anal cancer samples (76.5%), followed by A2, which was identified in 20/119 (16.8%) of samples. A4 was detected in 5/119 samples (4.2%). Two samples were classified as B1 (1.7%), one as A3 (0.8%) and one as D1 (0.8%). Further detail of HPV 16 sub-lineages in anal cancers is described in detail in Table 2 and Figure 1.

**Table 2.** HPV 16 sub-lineages identified in the anal cancer and asymptomatic cohorts.



**Table 2.** *Cont.*

0.002

**Figure 1.** Phylogenetic tree representing the HPV 16 sub-lineages present in the anal sample and control groups. **Figure 1.** Phylogenetic tree representing the HPV 16 sub-lineages present in the anal sample and control groups.

A2, identified in 23/134 (17.2%) of samples. D1 sub-lineage was identified in 4/134 samples (3.0%), and C1 and B1 were identified in two cases each (1.5%). B2 was present in 1/134 (0.7%). Table 2 describes the number of cases identified for each sub-lineage, and Figure

*3.3. Differences in Prevalence of HPV 16 Sub-Lineages between Anal Cancer and Control Cohort*

No major differences in the proportion of A1 and A2 sub-lineages were observed between the case vs. control cohorts, being 76.4% vs. 76.0% and 16.2% vs. 17.0%, respectively. Whereas the case cohort revealed the presence of the A4 sub-lineage in 4.2% of anal cancers, this sub-lineage was not present in the control cohort. Conversely, the C lineage was present in 1.5% of control specimens and was not detected in cancer cases. Finally, there was a slight increase in the D1 sub-lineage observed in the control vs. case cohort

1 contains the phylogenetic tree obtained from the control cohort.

*3.2. HPV 16 Sub-Lineages in the Control Cohort*

(3.0% vs. 0.8%).
