Guidelines for Use of the Spike-In Control Mixes
The DriverMap TCR/TCR RNA Spike-in controls have been validated using Cellecta’s DriverMap™ AIR TCR-BCR Profiling Kit. The AIR assay is a multiplex PCR-based NGS assay that quantitatively profiles all functional immune receptor isoforms, i.e., TCR chains (TRA, TRB, TRD, TRG) and BCR chains (IGH, IGK, and IGL). With the AIR assay, you can profile the CDR3 region or full-length receptor regions (see Fig. 2). For more information on the DriverMap AIR Assay, please visit Cellecta’s website. When using AIR-seq assays different than the DriverMap AIR assay, the efficiency of Spike-in controls detection may vary so some adjustments to the protocol may be necessary.
The AIR TCR and BCR Spike-In Control Mixes contain 3 different construct variants of each of the 13 TCR isoforms or 16 BCR isoforms, respectively. The 3 variants of each isoform are present at 250, 1000, and 4000 molecules per microliter. As a result, 2 µl of the mix contains 500, 2000, and 8000 theoretical molecules of each isoform variant. These 16-fold differences in concentrations enable a quantitative estimation in a 16-fold dynamic range of the absolute number of molecules of each TCR or BCR isoform in the profiled sample.
It is also important to include in each experiment the PBMC RNA reference standard provided in the kit. Cellecta’s PBMC control RNA sample acts as a universal control (with Spike-in Control mix) to measure and sometimes compensate for batch effects in experiments. Using the same reference universal positive control RNA sample in different experiments is the best strategy to evaluate and potentially compensate for batch effects due to differences in assay conditions.
To accurately measure the number of control constructs in the DriverMap AIR assay, it is recommended to sequence at a higher depth (appr. 5-10M reads per sample) to achieve an average of at least 10-20 per cDNA molecule. Sequencing at a lower depth could reduce control constructs’ measured copy number.
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