Eukaryotic tRNAs contain on average 14 modified nucleotides per molecule. Modifications in tRNAs are responsible for their structure and their function. During stress, tRNAs have been shown to modulate and reprogram their tRNA status in order to fine-tune translational cellular responses. tRNAs deficient in even one modification have been known to cause deleterious health effects for organisms. However, full scale investigations thus far into the global biological functions of many tRNA modifications have been insufficient in scope due to the difficulty of resolution of tRNA isodecoders. Also, in order to obtain exact information regarding modifications, tools thus far used to study tRNA modifications are at best imprecise for modification fraction determination. However, in the advent of high-throughput sequencing, it is possible to determine and quantify modifications in RNA molecules. Herein, the method of tRNA-seq and application to human eukaryotic tRNA is described. Modifications impact and impair the process of reverse transcription, and due to this nuclear-encoded and mitochondrial Watson-Crick interface modifications can be identified via proxy through sequencing due to the presence of mutations and truncations in cDNA. These mutations and stops in the cDNA reads allow us to assign a metric for each position in all eukaryotic tRNAs, which we call modification index (MI). From MI, we can determine positions of modification by proxy. Using pre-existing knowledge of tRNA modifications from homology and other studies, we are able to validate existing modifications as well as discover new sites. Further bioinformatic work is done to identify modification signatures based on context, modification identity, and reverse transcriptase. This is used to identify other types of modifications more broadly in other RNA molecules.