<i>k</i>-means clustering of CaCx cases portraying purely integrated HPV16 genomes.

<p> <b><i>Cluster 1</i></b><i>: samples with low viral load, low E7 expression; <b>Cluster 2:</b> samples with high viral load, low E7 expression; <b>Cluster 3:</b> samples with moderate viral load, moderate E7 expression; <b>Cluster 4:</b> samples with low to moderate viral load, high E7 expression.</i></p

Methylation analysis.

<p>(<b>A</b>) Bar diagram comparing percentage of samples methylated at different CpGs within the LCR of HPV16 genome between <i>E2</i>-intact and <i>E2</i>-disrupted samples. (<b>B</b>) Diagrammatic representation of the mentioned CpG positions within the LCR: 7862 – Viral replication origin; 31 – SpI binding site; 37 and 43 – E2BS II; 52 and 58 – E2BS I.</p

APOT-coupled-quantitative-RT-PCR assay.

<p>(<b>A</b>) Representative gel electrophoresis showing P2 - (dT)17-P3 products. Lane 1: sample showing presence of 1050 bp band-size indicating episomal viral genome. Presence of other band-sizes indicates concomitant status; Lanes 2, 3, 4, 6: absence of band-size 1050 bp indicates integrated status of these samples. Lane 5: 100 bp ladder (Roche)<b>.</b> (<b>B</b>) TaqMan-based quantitative RT-PCR amplification plots of <i>E7</i> and <i>E4</i> cDNA. <i>E7</i> is transcribed in both episomal (pure or concomitant) and integrated cases, but <i>E4</i> is transcribed in only episomal cases. (<b>C</b>) Agarose gel electrophoresis of PCR products of <i>GAPDH</i> cDNA (primers span exon-exon junction). Lane 1: negative control; Lane 2: CaSki cell line DNA (negative control); Lanes 3–7: sample cDNAs showing specific band size of 106 bp; Lanes 3 and 7 show episomal (pure or concomitant) samples T323 and T329, respectively, while, Lanes 4–6 show purely integrated samples T327, T326 and T328, respectively; Lane 8: <i>Hae</i> III digested ?x174 DNA marker (Promega). (<b>D</b>) Agarose gel electrophoresis of PCR products of <i>TP53</i> cDNA (primers span introns). Lane 1–6, 8, 9: sample cDNA not showing specific band size of 448 bp; Lanes 1, 2, 4, 6 and 9 show purely integrated samples T326, T327, T345, T344 and T328, respectively, while, lanes 3, 5 and 8 show episomal (pure or concomitant) samples T329, T323 and T339, respectively; Lane 10: CaSki cell line DNA (positive control); Lane 11: sample DNA (positive control); Lane 12: water (negative control); Lane 7: <i>Hae</i> III digested ?x174 DNA marker (Promega).</p

Classification of samples with intact <i>E2</i> gene into those harbouring pure episomal and concomitant (episomal+integrated) viral genomes based on <i>E7</i> C_T/<i>E4</i> C_T derived from APOT-coupled-quantitative-RT-PCR assay.

<p>Concomitant refers to the presence of both episomal and integrated viral genomes.</p

Samples with purely integrated viral genomes grouped into 4 clusters after <i>k</i>-means clustering analysis.

<p>Cluster 1: low viral load and low <i>E7</i> expression; Cluster 2: high viral load and low <i>E7</i> expression; Cluster 3: moderate viral load and moderate <i>E7</i> expression; Cluster 4: low to moderate viral load and high <i>E7</i> expression.</p

Linear regression analyses.

<p>(<b>A</b>) Correlation of <i>E7</i> C_T/<i>ACTB</i> C_T with viral load (natural log values) in CaCx cases with episomal (purely episomal and concomitant) viral genomes; (<b>B</b>) Correlation of <i>E7</i> C_T/<i>ACTB</i> C_T with viral load (natural log values) in CaCx cases with integrated viral genomes; (<b>C</b>) Correlation of <i>E2</i> C_T/<i>ACTB</i> C_T with viral load (natural log values) with respect to <i>E2</i> gene in CaCx cases with episomal (purely episomal and concomitant) viral genomes.</p

Final cluster centers obtained from <i>k</i>-means clustering of purely integrated samples.

*<p> <i>E7 C_T was derived from APOT-coupled-quantitative-RT-PCR assay.</i></p>†<p> <i>viral load values were transformed to natural log values.</i></p