Abstract
High-throughput sequencing of the products of 5′ RNA ligase-mediated rapid amplification of cDNA ends (5′ RLM-RACE) reactions (RACE-SEQ) enables the mapping and digital enumeration of expected and novel 5′ ends in RNA molecules. The resulting data are essential in documenting the mechanism of action and precision of endonucleolytically active, RNA-targeting drugs such as RNase H-active antisense or small interfering RNA. When applied to error-prone replication systems such as RNA viruses or in vitro RNA replicon systems, the method can additionally report the relative susceptibility of known and unknown polymorphisms to a prospective sequence-specific drug, making it a powerful tool in patient selection and stratification as well as resistance prediction.
We describe the preparation of sequencing libraries for ultra-high depth 5′ RLM-RACE analysis on two popular second-generation high-throughput sequencing platforms (Illumina, Ion Torrent) and supply a detailed bioinformatics analysis pipeline for target site activity definition and enumeration. We further illustrate how the pipeline can be simply modified to generate polymorphism-specific drug susceptibility data from in vitro replicon experiments (RACE-SEQ-MM), in a patient-free manner, to cover both known and unknown target site variants in the population.
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Usher, L., Theotokis, P.I., Moschos, S.A. (2019). RACE-SEQ and Population-Wide Polymorphism Susceptibility Testing for Endonucleolytically Active, RNA-Targeting Therapeutics. In: Gissberg, O., Zain, R., Lundin, K. (eds) Oligonucleotide-Based Therapies. Methods in Molecular Biology, vol 2036. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9670-4_17
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DOI: https://doi.org/10.1007/978-1-4939-9670-4_17
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