The use of non-functional clonotypes as a natural spike-in for multiplex PCR bias correction in immune receptor repertoire profiling

High-throughput sequencing of immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful methods for TCR/BCR repertoire reconstruction and analysis have been developed in the past decade. However, detection and correction of the discrepancy between real and experimentally observed lymphocyte clone frequencies are still challenging. Here we formulated a quantitative measure of V- and J-genes frequency bias driven by multiplex PCR during library preparation called Over Amplification Rate (OAR). Based on OAR concept, we developed an original software for multiplex PCR-specific bias evaluation and correction named iROAR: Immune Repertoire Over Amplification Removal (https://github.com/smiranast/iROAR). The iROAR algorithm was successfully tested on previously published TCR repertoires obtained using both 5’ RACE (Rapid Amplification of cDNA Ends)-based and multiplex PCR-based approaches. The developed tool can be used to increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.