Targeted TCR-CDR3 Sequencing from Tumor, Normal, and PBMC

Genomic DNA was isolated from the tumor, NAT and PBMC single cell suspensions using QIAamp blood mini kits (Qiagen, Hilden, Germany). Targeted TRB-complementarity determining region 3 (TRB-CDR3) libraries were constructed using the ImmunoSEQ hsTCRB v3.0 kit (Adaptive Biotechnologies, Seattle, WA) on 1µg of genomic DNA isolated from the biospecimens according to the manufacturer’s protocol. Briefly, bias-controlled multiplex PCR was applied to amplify all possible rearranged genomic TCRβ sequences using an equimolar pool of the 45 TCR Vβ forward primers, and an equimolar pool of the TCR Jβ reverse primers. The following thermal cycling conditions were used for amplification: 1 cycle at 95°C for 15 minutes, 25 to 40 cycles at 94°C for 30 seconds, 59°C for 30 seconds, and 72°C for 1 minute, followed by 1 cycle at 72°C for 10 minutes (15 (link)). The final PCR products at 200bp length were pooled and sequenced at survey level resolution on the Illumina MiSeq platform (v3 150 cycle) in the Genomics Core Facility at the Fred Hutchinson Cancer Research Center as previously described (16 (link), 17 (link)).