RACE-SEQ and Population-Wide Polymorphism Susceptibility Testing for Endonucleolytically Active, RNA-Targeting Therapeutics

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2036; p. 283
• Main Authors: Usher, Louise, Theotokis, Pantazis I, Moschos, Sterghios A
• Format: Journal Article
• Language: English
• Published: United States 2019
• Subjects: Computational Biology - methods; DNA, Complementary; Genetic Therapy - methods; High-Throughput Nucleotide Sequencing - methods; Humans; MicroRNAs - genetics; Nucleic Acid Amplification Techniques; Polymorphism, Genetic; RNA; RNA Interference; RNAi; RACE; RACE-SEQ-MM; Antisense; siRNA; 5′ RLM-RACE; Mechanism of action; Pipeline; RACE-SEQ
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-9670-4_17

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.