The integration of exogenous DNA into the human genome can cause somatic mutations associated with oncogenesis. For example, the insertion of HPV DNA into human chromosomes is the single most important event leading to tumorigenesis in cervical cancer. It is also now preventable with vaccines against HPV. In contrast to viral DNA integrations, the instances and repercussions of bacterial DNA integration into the somatic human genome are less clear. Yet bacterial DNA integrations found to be associated with tumorigenesis could also be prevented using therapeutics like vaccines that limit exposure to the bacteria. This proposal has three objectives aimed at addressing our gap in knowledge about bacterial DNA integrations. First, virtual machines will be developed for LGTSeek and LGTview, our bioinformatics tools that we have used previously to detect bacterial DNA integrations in human genome sequencing projects. These virtual machines would enable these tools to be run by a wide variety of users, from the bioinformatically savvy to the nave. This provides a resource to the community to enable detection of such integrations by a wider variety of scientists. In addition, LGTSeek and LGTView will be used to further interrogate publicly available cancer genome data where such integrations are likely to occur because the tissues are exposed to the microbiome (e.g. colon). Second, genome and transcriptome sequencing will be undertaken of new stomach adenocarcinoma samples and acute myeloid leukemia samples. This objective is aimed at reproducing previous results that suggest the presence of bacterial DNA integrations. These sequencing efforts would include control samples with exogenous bacterial nucleic acids added to the sample in order to quantify the formation of chimeras in modern sequencing techniques. Third, the effect that previously detected bacterial DNA integrations have on transcription will be interrogated using luciferase reporter constructs. Integrations that lead to up-regulation of the gene will be further interrogated by reconstructing the integrations in cells using the CRISPR/Cas9 system. Collectively, this research is expected to improve our understanding of the extent and significance of bacterial DNA integrations in the somatic human genome.
The integration of bacterial DNA in the human somatic genome could result in diseases due to insertional mutagenesis. The experiments proposed here aim at developing tools for the research community to detect such integrations, assessing the prevalence of such integrations in cohorts of gastric cancer and acute myeloid leukemia patients, and testing the consequence of known integrations on transcript abundance and phenotype.
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