iROAR

iROAR evaluates and corrects multiplex PCR amplification bias in high-throughput sequencing of T-cell receptor (TCR) and B-cell receptor (BCR) repertoires.

Key Features:

• Bias Evaluation: Identifies discrepancies between read count and clone count-based frequencies of non-functional clonotypes to detect multiplex PCR-specific biases.
• Quantitative Measure - Over Amplification Rate (OAR): Calculates the Over Amplification Rate (OAR) to quantify V- and J-gene frequency bias induced by multiplex PCR during library preparation.
• Bias Correction: Applies an algorithmic correction to adjust clonal counts in immune repertoire output tables, including tables produced for VDJtools.
• Validation and Testing: Validated on previously published TCR repertoires obtained via 5' RACE-based and multiplex PCR-based approaches and benchmarked against a biological spike-in method for PCR bias evaluation.

Scientific Applications:

• Comparative Analysis: Enables more accurate comparison of immune repertoires reconstructed by different experimental methods by correcting amplification bias.
• Adaptive Immunity Studies: Provides corrected clonal frequency data for investigations of adaptive immune responses using TCR and BCR sequencing.
• Clinical Research: Improves the precision of repertoire-based analyses relevant to disease monitoring and diagnostics, including cancer and autoimmune disorders.

Methodology:

iROAR detects anomalies in the ratio between read count and clone count-based frequencies of non-functional clonotypes, calculates OAR for V- and J-genes, and applies an algorithmic correction to adjust clonal counts in repertoire output tables (e.g., VDJtools).