Complete multi-chain adaptome amplification from whole blood and PBMC via novel dam-PCR technology

Abstract Profiling the immune repertoire through NGS allows detailed, sequence-specific insight into the adaptive immune response. One of the key challenges during immune receptor amplification is the formation of dimers, which can compete with the immune amplicons of interest during library preparation. Therefore, we developed a novel PCR technique, dimer-avoided multiplex PCR (dam-PCR), that effectively avoids primer dimer formation and allows for the all-in-one qualitative amplification of all BCR and TCR receptors in a single, quantitative reaction. Here, we present this technology’s capabilities of bias-free immune repertoire amplification, even for low frequency CDR3s. First, we demonstrate the ability of this method to accurately detect a Jurkat cell line that was spiked into pre-screened sorted donor CD4+ cells at 1%, 0.1%, 0.01%, 0.001%, and 0.0001%. RNA from each spike-in percentage was extracted and amplified in duplicate using our standard amplification protocol to assess repeatability of CDR3 discovery between replicates, as well as establish the limits of detection and quantitation. Then, we demonstrate the method’s ability to amplify a synthetic repertoire, created by spiking RNA from two individuals of known repertoire into each other at 75%, 50%, and 25%. The mixed RNA, as well as pure RNA from each individual, was then amplified to assess correlation between the predicted repertoire and amplified repertoire at each spike-in percentage. For both the Jurkat cell level spike-in test, as well as the RNA level spike-in test, the amplified repertoire was successfully predicted at an R2 value of > 0.99. This single reaction method will allow for a cost effective, all-inclusive, and quantitative analysis of immune repertoires.