Figure 3.

Detection of HPV mRNA in Case 2 by targeted sequencing. Total RNA was first extracted from the Case 2 brain metastasis, followed by separation of the mRNA fraction using oligo dT magnetic beads. This was followed by cDNA library preparation and enrichment of HPV encoded cDNAs using the SureSelect protocols. After Illumina sequencing, reads were aligned to HPV16 reference genome (NC_001526.4) and visualized by IGV software. (A) Data revealed a high level of E6 and E7 expression in the tumor. (B) The junction track showing alternative splice junctions in the E6 gene. The E6*I splice junction had more coverage (8368 reads) than E6*II (1561 reads). (C) Consistent with the DNA sequencing, human reads were detected for the integration site at 8q24.21, indicating the presence of a chimeric mRNA encoded from both HPV16 and the host genome.