High-throughput Immune Repertoire Profiling

The TRB and IGH genes containing the entire V, D, and J gene segments were amplified with TRB-A and –B and IGH primer pools, respectively, from 250 ng PB genomic DNA, which corresponds to ~37,500 genome equivalents (24 (link)). Compared to our previously published protocol, the TRB PCR assay was refined by using a touch-down PCR protocol and a mixed primer TRB-A/B tube. In two consecutive PCR reactions amplicons were tagged with Illumina adapters and indices as previously described (25 (link), 26 (link)). PCRs were performed using Phusion HS II (Thermo Fisher Scientific Inc., Germany). Amplicons were purified after agarose gel electrophoresis using the NucleoSpin^® Gel and PCR Clean-up kit (Macherey-Nagel, Germany). Before being subjected to NGS the concentration and quality of the amplicons/libraries was determined using Qubit (QIAGEN, Germany) and Agilent 2100 Bioanalyzer (Agilent technologies, Germany), respectively.