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154 Universal synthetic spike-in controls for accurate adaptive immune receptor profiling
  1. Alex Chenchik,
  2. Tianbing Liu,
  3. Mikhail Makhanov,
  4. Dongfang Hu and
  5. Paul Diehl
  1. Cellecta, Inc., Mountain View, CA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background The results of adaptive immune receptor (AIR) repertoire diversity assays can be affected by various biases from differences in conditions in the RT-PCR and NGS sequencing steps. Spike-in synthetic controls can be used as calibration standards to address these biases

Methods In this study, we synthesized near full-length BCR and TCR constructs that mimic seven different IGH, IGK, IGL, TRB, TRA, TRG and TRD genes. To test our spike-in controls, three sets of variants at different concentrations were added to the RNA samples before the reverse transcription reaction with Cellecta’s DriverMap™ Adaptive Immune Receptor (AIR) Profiling Assay. The DriverMap™ protocol uses reverse gene-specific primers with unique molecular identifiers (UMI), allowing UMI-based correction of amplification biases during data analysis

Results Calculating UMI-based correction and comparing that with spike-in controls enabled us to differentiate between real and background sequences and estimate the average sequencing error rate at 0.4%-0.8% per base, which is within the reported range of Illumina sequencing.

Conclusions This suggests that our spike-in controls are reliable and may be used as a universal tool to correct AIR protocol biases and calculate error and mutation rates in different AIR profiling assays.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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