Strategy for PCR amplification, hybridization, and sequencing of rearranged TCRβ CDR3 regions. A generic rearranged TCRβ CDR3 region PCR product is shown, indicating the constituent Vβ segment, Dβ segment, Jβ segment, and the nontemplated nucleotides inserted at the Vβ-Dβ and Dβ-Jβ junctions. Universal adapter sequences that permit solid-phase PCR on the Illumina Genome Analyzer Cluster Station (GA F and GA R) are incorporated into the 5′ and 3′ ends of the PCR products that capture each rearranged TCRβ CDR3 region. Forty-five forward primers were designed, each specific to a single functional Vβ segment or a small family of Vβ segments. The 3′ end of each Vβ forward primer is anchored at position −43 in the Vβ segment, relative to the recombination signal sequence, thereby providing a unique Vβ tag sequence within the amplified region. Vβ forward primers were designed for all known nonpseudogenes in the TCRβ locus. The 13 reverse primers specific to each Jβ segment are anchored in the 3′ intron, with the 3′ end of each primer crossing the intron/exon junction. The Jβ reverse primers were designed to be anchored at their 3′ ends on a consensus splice site motif to minimize overlap with the sequencing primers. Thirteen sequencing primers were designed that are complementary to the amplified portion of the Jβ segment, such that the first few bases of sequence generated will capture the unique Jβ tag sequence. The sequencing primers were designed so that promiscuous priming of a sequencing reaction for one J segment by a primer specific to another J segment would generate sequence data starting at exactly the same nucleotide as sequence data from the correct sequencing primer.