Highly accurate RNA and DNA sequencing: Application to longstanding questions in aging and cancer
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Accurate DNA replication and RNA transcription are critically important for proper cell functioning: the fidelity of these processes is crucial; infidelity can lead to cellular dysfunction and disease. The key problem in studying the fidelity of these processes is the accurate detection of rare DNA and RNA mutations, which result as a consequence of infidelity. Until recently, this has not been possible, as the high error rates of available methods has limited their ability accurately detect rare mutations among a preponderance of wildtype molecules. The solution to this problem, as the Loeb lab and others have found, is to perform single molecule sequencing of individually barcoded DNA and RNA molecules. In the present work, I present three projects which apply the use of barcoding individual DNA and RNA molecules in order to enable highly accurate and sensitive analyses of DNA replication and RNA transcription fidelity. (i) The question of why CS patients don’t get cancer despite being repair-deficient has puzzled scientists for decades. While many have speculated as to the cause, we have applied Duplex Sequencing to definitively answer this question: CS patients fail to develop cancer because they do not accumulate mutations more quickly than repair-proficient individuals. In addition to finally solving this long-standing mystery, we provide novel insights into the mutagenic consequences of UV treatment in CS cells, at an unparalleled sensitivity. (ii) The question of why GBM patients do so poorly and always recur has long plagued doctors and scientists. Here, we expand on the excellent clonal mutation work of our predecessors, revealing that the substantial inter- and intra-tumoral clonal heterogeneity is further compounded by considerable subclonal heterogeneity. We show that subclonal mutations are highly heterogenous within individual GBM tumors, between GBM tumors from different patients, as well as between primary and recurrent tumors from the same patient. Our findings of high subclonal heterogeneity in GBM tumors suggest that GBM patients do so poorly because their tumors already contain a reservoir of mutations that potentially enable them to adapt to any treatment currently available. This underlies the importance of expanding subclonal mutation studies of GBMs to better understand their mutational makeup. (iii) The question of what, if any, contribution RNA mutations have to health and disease has been one that has remained unanswered for more than 50 years. RNA mutations have long been hypothesized to play roles in human health and disease, as well as in several other processes, including RNA virus evolution and bacterial resistance to antibiotics. Unfortunately, until now, it has been very difficult to study the hypothesis that transcriptional mutagenesis, resulting in RNA mutations, contributes to or drives these processes because there have not been the tools available to do so. I have, therefore, developed a method to accurately sequence RNAs. Here, I demonstrate that Accurate RNA Consensus Sequencing (ARC-seq) has inherent adaptability to enable increased stringency, which eliminates a high level of damage-induced artifacts. I also show that RNA polymerase mutants induce increased transcriptional mutagenesis in vivo, with different mutants producing varying RNA mutation spectrums. Finally, I demonstrate the utility of ARC-seq to address questions on the biological importance of transcriptional mutagenesis in vivo by using ARC-seq to show that oxidative stress induces high levels of transcriptional mutagenesis in both mRNA and rRNA. Thus, ARC-seq will enable studies on how perturbing a cell’s environment or machinery affects the fidelity of transcription and to what extent RNA mutations contribute to aging, cancer, and neurodegeneration, as well as the evolution and acquired resistance of viruses and bacteria. Together the three projects encompassed in this thesis demonstrate the power of combining the use of barcoding individual DNA and RNA molecules in order to enable highly accurate and sensitive analyses of DNA replication and RNA transcription fidelity.