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High-throughput RNA sequencing was developed almost a decade ago. Used originally to map the transcriptome to single-nucleotide resolution, advancements have expanded this powerful method to investigate many different aspects of cellular RNA biology. Along with changes in transcriptome in response to a multitude of different conditions, the DNA-RNA, RNA-RNA, and RNA-protein interactions have been defined. Additionally, RNA high throughput sequencing has been used to study differential splicing, structural changes in cellular RNA, induction of transcription, RNA editing, rates of translation, and many other biological parameters.,Here, three additional methods are presented that expand the functionalities of RNA high-throughput sequencing. First, we developed a method to determine tRNA aminoacylation levels via high-throughput sequencing. Previously, chemistry was used to distinguish between charged and uncharged tRNA using microarray methods. We harness the previously established chemistry to develop a one-pot sequencing method to determine the tRNA aminoacylation levels in mammalian cells. Next, we report a method to detect modifications in tRNAs using deep sequencing. By defining the combination of mutation rate and stop rate at each site as the 'modification index', we identify likely sites of modification. Then, based on the effect of treatment with a demethylase and the contributions of the stop and mutation rate to the modification index, the identity of the modification can be discerned. Finally, we work towards developing a method to predict sites of methylation in tRNA and mRNA by high-throughput sequencing and machine learning. Using model oligonucleotides to explore the effect of sequence context on the modification index of a given RNA methylation, we define the 'modification signature' for six different modifications. This modification signature can be used to identify the position, identity, and abundance of modifications in tRNA with high accuracy, with the possible expansion into mRNA modifications. These three methods add to the already expansive list of methods to interrogate RNA biology and can be used to further our knowledge of the importance of RNA modifications in cellular biology


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