The Flux Capacitor

The Flux Capacitor predicts transcript molecule abundances and alternative splicing events from RNA sequencing (RNA-Seq) experiments to quantify expressed RNAs and characterize technical biases in cDNA library workflows.

Key Features:

• Bias Analysis and Mitigation: Systematically analyzes biases across experimental steps including reverse transcription, fragmentation, adapter ligation, PCR amplification, gel segregation, and sequencing.
• Model Development: Develops universally applicable, parameterized models for each identified RNA-Seq step using empirical attributes of specific experimental protocols.
• Simulation Pipeline - The Flux Simulator: Implements the models in the Flux Simulator to generate read distributions that replicate those observed in actual experiments.
• Insight into Hidden Precursors: Simulates read generation along gene bodies to reveal hidden precursor effects that influence final read configurations and produce compensatory or enhancing effects.
• Identification of Systematic Biases: Identifies sources of systematic bias, including biases arising from RNA hydrolysis used in common fragmentation protocols.

Scientific Applications:

• Transcript Abundance Quantification: Improves quantitative prediction of transcript molecule abundances from RNA-Seq read data.
• Alternative Splicing Analysis: Supports detection and interpretation of alternative splicing events by accounting for protocol-dependent read distribution biases.
• Protocol Evaluation: Enables assessment of how combinations of technical steps influence sequencing outcomes to inform experimental protocol evaluation.

Methodology:

The approach decomposes RNA-Seq experiments into technical components (reverse transcription, fragmentation, adapter ligation, PCR amplification, gel segregation, sequencing), develops parameterized models for each step using empirical protocol attributes, and integrates those models in the Flux Simulator to generate read distributions that replicate observed experiments.