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.