Multiplex PCR for Amplification of TRB Locus

The rearranged V, D, and J gene segments of the TRB locus were amplified together in a multiplex PCR using TRB-A/-B primer pools and 250 ng of genomic DNA [33 (link)]. The primers were purchased from Metabion International AG (Martinsried, Germany). As described in Schliffke et al. [34 (link)], two consecutive PCR reactions were performed to generate TRB fragments tagged with Illumina-compatible adapters for hybridization to the flow cell and seven nucleotide barcodes for sample identification. All PCRs were performed using Phusion HS II (Thermo Fisher Scientific Inc., Darmstadt, Germany). After gel electrophoretic separation, TRB amplicons were purified using the NucleoSpin^® Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany), quantified on the Qubit platform (QIAGEN, Hilden, Germany), and pooled to a final concentration of 8 nM. The quality of the TRB amplicon pools was controlled on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany) before undergoing NGS. The samples were sequenced with a mean sequencing depth of 80,520 reads (range 42,440–137,852 reads).
NGS and demultiplexing was performed on an Illumina MiSeq sequencer (600-cycle single indexed, paired-end run, V3-chemistry). Analysis of the rearranged TRB loci was computed and plotted as previously described [35 (link), 36 (link)].