Synthetic TCR/BCR RNA spike-in controls for assessing immune receptor profiling assay accuracy

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Abstract Synthetic TCR and BCR RNA spike-in controls can be used as universal standards to account for biases caused by PCR and NGS steps of adaptive immune receptor (AIR) repertoire profiling assays. We designed 48 BCR and 39 TCR mRNA synthetic constructs to cover nearly full-length V(D)JC structures, which represent all seven TCR (TRB, TRA, TRG, TRD) and BCR (IGH, IGK, and IGL) hypervariable chains. To identify cross-sample contamination, we spiked in 2 ul of BCR (16x3) and TCR (13x3) triplex isoform pools and detected the presence of the sequence in the sample. Furthermore, we integrated 48 BCR and 39 TCR premixed controls which are mixed in a 16:4:1 ratio into Cellecta’s DriverMap™ Adaptive Immune Receptor Profiling Assay, which employs a gene-specific, multiplex RT-PCR method with unique molecular indices (UMIs). The spike-in controls coupled with UMI-based PCR amplification can help distinguish between control and background sequences. We observed a consistent linear relationship between the concentration of spike-in controls and their molecular counts, with an average sequencing error rate between 0.4%-0.8% per base, which is in line with Illumina sequencing standards. These findings validate the effectiveness of our synthetic spike-in controls in correcting biases in AIR protocols, offering a reliable estimation of error and mutation rates in the DriverMap AIR assay or similar NGS-based immune receptor profiling methods.

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