RACE-SEQ

RACE-SEQ maps and digitally enumerates 5' RNA ends from 5' RLM-RACE sequencing to characterize endonucleolytic activity and polymorphism susceptibility of RNA-targeting therapeutics.

Key Features:

• 5' RLM-RACE compatibility: Processes data derived from 5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) experiments for end mapping.
• Precise 5' end mapping and digital enumeration: Identifies and counts both expected and novel 5' RNA ends at high resolution.
• High-depth sequencing support: Optimized for ultra-high depth second-generation platforms including Illumina and Ion Torrent.
• Therapeutic mechanism characterization: Enables documentation of mechanism and precision of endonucleolytically active, RNA-targeting therapeutics such as RNase H-active antisense oligonucleotides and small interfering RNAs (siRNAs).
• Polymorphism susceptibility analysis: Reports relative susceptibility of known and unknown polymorphisms, applicable to error-prone replication systems such as RNA viruses and in vitro RNA replicon systems.
• Modular analysis pipeline (RACE-SEQ-MM): Analysis pipeline can be modified to generate polymorphism-specific drug susceptibility data from in vitro replicon experiments (RACE-SEQ-MM).
• R-based analysis script: Provides an R script (RACE-SEQ-lite) for bioinformatics analysis with automatic installation and loading of required R packages.

Scientific Applications:

• Mechanism-of-action mapping: Characterizes cleavage sites and precision of RNase H-active antisense oligonucleotides and siRNAs by mapping 5' ends.
• Resistance and susceptibility profiling: Assesses relative susceptibility of target-site polymorphisms to sequence-specific drugs to inform resistance prediction.
• Viral and replicon variant analysis: Detects and quantifies polymorphisms in error-prone RNA viruses and in vitro RNA replicon systems to evaluate variant-specific drug effects.
• In vitro polymorphism-specific susceptibility assays: Generates polymorphism-specific drug susceptibility data from in vitro replicon experiments without requiring patient-derived samples (RACE-SEQ-MM).
• Patient stratification support: Provides data to inform patient selection and stratification based on target-site variant susceptibility.

Methodology:

Analysis is performed with the R script RACE-SEQ-lite run under R version ≥ 3.4 with required R packages automatically installed and loaded; the provided bioinformatics analysis pipeline defines and enumerates target-site activity and can be modified to produce RACE-SEQ-MM polymorphism-specific susceptibility outputs.