Using Sequencing Approaches to Characterize Tumor Microbial Communities and Viral Transcriptomes

Abstract

Microorganisms are the etiologic agents of many human diseases. Next-generation sequencing (NSG) allows for sensitive characterization of nucleic acids and offers the ability to identify and characterize microorganisms present in human disease. In the context of cancer, NGS approaches such as whole-genome sequencing of DNA (WGS) and RNA sequencing (RNAseq) can be used to identify biologically relevant bacteria and viruses that are present in tumor tissue. In the context of viral infections, RNAseq provides the ability to profile the RNA transcripts produced by viruses and allows for the identification of uncharacterized viral transcripts.

Human cancers are associated with viruses and bacteria that can contribute to carcinogenesis, and NGS approaches can be used to identify unknown interactions between cancers and microorganisms. We hypothesized that a virus could be associated with blastic plasmacytoid dendritic cell neoplasm (BPDCN), a cancer thought to arise from plasmacytoid dendritic cells which are crucial sensors of viral infection. We developed a computational virus discovery approach that, while capable of identifying the etiologic virus of other cancers in a blinded manner, did not identify a virus associated with BPDCN. Next, we hypothesized that geographic patterns in the incidence of esophageal squamous cancer (ESCC) may be explained by tumor-associated bacteria. We found that bacterial genera such as Fusobacterium which are associated with the carcinogenesis of other gastrointestinal cancers are present at high relative abundance in ESCC tumors from patients around the world. Furthermore, we identified a strong association between the oral and tumor bacterial communities of ESCC patients from Tanzania, raising the possibility that oral microbial communities could be associated with carcinogenesis in this context.

We used short- and long-read RNAseq to study viral transcripts, with the hypothesis that uncharacterized transcripts of biological relevance are produced during viral infections. First, we studied the RNAs generated by polyomaviruses, a family of viruses associated with devastating diseases including cancer. We identified novel transcripts, including “wraparound transcripts” which arise from pre-mRNAs that circle the viral genome multiple times. One such wraparound transcript encodes a novel oncoprotein, superT, which we find in human cancer. Next, we investigated the coronavirus SARS-CoV-2 which has driven the COVID-19 pandemic. We found that SARS-CoV-2 generates non-canonical subgenomic RNAs (sgRNAs) that increase in abundance over time during infection and are not generated in the homology-dependent manner canonical sgRNAs are produced. We find evidence that these non-canonical sgRNAs may influence the coding capacity of SARS-CoV-2.

Together, my research uses NGS approaches to provide insight into relationship between viruses, bacteria, and cancers and vastly expands the known transcript diversity of viruses that are relevant to human diseases.