RT Journal Article SR Electronic T1 The use of non-functional clonotypes as a natural calibrator for quantitative bias correction in adaptive immune receptor repertoire profiling JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.03.24.436794 DO 10.1101/2021.03.24.436794 A1 Anastasia Smirnova A1 Anna Miroshnichenkova A1 Yulia Olshanskaya A1 Michael Maschan A1 Yuri Lebedev A1 Dmitriy Chudakov A1 Ilgar Mamedov A1 Alexander Komkov YR 2022 UL http://biorxiv.org/content/early/2022/11/21/2021.03.24.436794.abstract AB High-throughput sequencing of adaptive immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful TCR/BCR repertoire reconstruction and analysis methods have been developed in the past decade. However, detecting and correcting the discrepancy between real and experimentally observed lymphocyte clone frequencies is still challenging. Here we discovered a hallmark anomaly in the ratio between read count and clone count-based frequencies of non-functional clonotypes in multiplex PCR-based immune repertoires. Calculating this anomaly, 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 the 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 and compared with a biological spike-in-based method for PCR bias evaluation. The developed approach can increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.Competing Interest StatementMiLaboratories LLC (USA) holds the rights on the TRA-specific oligonucleotide sequences used in this study.