Name	Sequence (5′–3′)	Target allele	T a (°C)	Size (bp)	Melt temp (°C)	Comments
PCR and sequencing primers
GH46F	CCGGATCCTTCGTGTCCCCACAGCACG	All	65	271	–	Erlich and Bugawan 1990
GH50R	CTCCCCAACCCCGTAGTTGTGTCTGCA	All	65	271	–	Erlich and Bugawan 1990
CyguDRB1_exon2_R1	CTCTCCTCTCCACAGTGAAGCTCTCA	All (exon 2) except Cygu‐DRB1*03	65	293		New reverse primer specific to C. gunnisoni.
CyguDRB1_exon2_R2	CTCTCCGCTCCACAGCGAAGCTCTCA	Cygu‐DRB1*03	65	293	–	New reverse primer specific to C. gunnisoni.
CyguDRB1exon3‐R2	AGACCAGGAGGTTGTGGTGCTG	All (exon 3)	65	334	–	For cDNA PCR and sequencing.
454 adapter‐A	cgtatcgcctccctcgcgccatcagNNNNNNNNNN	–	64	306	–	Tail for GH46F (with 10 bp indices).
454 adapter‐B	ctatgcgccttgccagcccgctcag	–	64	296	–	Tail for reverse primer (e.g., GH50R or CyguDRB1_exon2_R1 and R2).
Assays 1–4: Noncompetitive PCRs for all known CyguDRB1 alleles
PD1DRB_F	CGTTTCCTGGAGCAAGTTTCACATG	All	67	96	–	This forward primer can amplify all six alleles.
PD1DRB_R	CGAACTCCTCTCGGTTGTGG	Cygu‐DRB1*01 Cygu‐DRB1*05 Cygu‐DRB1*06	67	96	80–81	This reverse primer amplifies three highly similar alleles (01, 05, and 06).
PD2DRB_F2	GGAGCGGATACGGTTCCTAC	Cygu‐DRB1*02	67	48	73.5–74.5
PD2DRB_R	GACCTCCTCCCGGTTATAGAT	Cygu‐DRB1*02	67	48	73.5–74.5
PD3DRB_F2	TTTCCTGGAGCAAGGTTCACA	Cygu‐DRB1*03	67	102	80.75–81.75
PD3DRB_R2	AAAGCGCACGTACTCCTCCA	Cygu‐DRB1*03	67	102	80.75–81.75
PD4DRB_F2	GTTTCCTGGAGCAAGGTTTCAG	Cygu‐DRB1*04	67	183	86–87
PD4DRB_R2	GCGTCCTTCTGGCTGTTGA	Cygu‐DRB1*04	67	183	86–87
Assay 5: Noncompetitive PCRs to resolve allele 01
PD5DRB_F	CGTTTCCTGGAGCAAGTTTCACATG	All	65		–	Same sequence as primer PD1DRB_F above.
PD5DRB_R2_Anc	CCTCTCCACAGTGAAGCTCTCACCA	Cygu‐DRB1*01 (poor amp for 05, 06)	67	264	87–88	PCR with this reverse primer will amplify three highly similar alleles (01, 05, and 06). However, the C t value for allele 01 will occur 8–10 cycles earlier compared to a PCR using the alternate reverse primer (PD5DRB_R2_Der).
PD5DRB_R2_Der	TCTCCTCTCCACAGTGAAGCTCTCAAAC	Cygu‐DRB1*05 Cygu‐DRB1*06 (poor amp for 01)	67	267	87–88	PCR with this reverse primer will amplify three highly similar alleles (01, 05, and 06). However, the C t value for alleles 05 or 06 will occur 8–10 cycles earlier compared to a PCR using the alternate reverse primer (PD5DRB_R2_Anc).
Assay 6: Competitive PCR to resolve allele 05 versus 06
PD6DRB_164G_F_Anc	AGTACCGCGCGGTGAGAG	Cygu‐DRB1*01 Cygu‐DRB1*05	56	33	75.5–76.5 and 84–85	Double peaks observed in dissociation curve.
PD6DRB_164A_F_Der	gcccgcccgcccgcccAGTACCGCGCGGTGAGTA	Cygu‐DRB1*06	56	49	82–83
PD6DRB_R_Cons	CCGGCCGCCCCAGCT	All alleles (two SNPs in D allele)	56		–	Conserved reverse primer.