130-P: COMPLETE BI-DIRECTIONAL SEQUENCING OF HLA-DRB1 EXONS 2 AND 3

Abstract

Many HLA-DRB1 sequencing kits include exons 2 and 3, but most are unable provide complete bi-directional sequencing coverage for both exons 2 and 3 including the highly polymorphic first hypervariable region. Here we describe a strategy that provides full bi-directional sequencing coverage using single forward and reverse direction sequencing mixes. Exon 2 amplification primers were designed to target all DRB1 alleles within introns 1 and 2 for locus specific amplification. Exon 2 sequencing primers were nested within the amplification primer binding sites, but outside exon 2. Exon 3 amplification primers were tailed and located within introns 2 and 3. All amplification and sequencing primers were located outside of exons 2 and 3. To prove accuracy of design, approximately 275 well characterized samples were screened using this strategy followed by analysis with uTYPE® sequencing analysis software. All samples were concordant with known typing results with the exception 3 samples. The 3 discordant samples contained novel positions not previously described within exon 3. Robustness of design was tested by modifying amplification and sequencing parameters to challenge kit stability and primer specificity. All testing resulted in DRB1 locus specific sequences. In addition, a reproducibility study was conducted where two operators tested 4 samples 4 times over 5 non-consecutive days. All results were 100% concordant. Complete exon 2 sequencing enabled resolution of at least 46 additional alleles when compared to current SeCore DRB1 kit design. Incorporation of exon 3 further discriminated DRB1∗1454 from 140101; DRB1∗120101 from ∗1206/17; DRB1∗13:01:01 from DRB1∗13:117; DRB1∗11:01:01 from DRB1∗11:01:08/97 and DRB1∗01:01:01 from DRB1∗01:50. The new SeCore® DRB1 kit has proven accuracy and reproducibility with the capability of novel allele discrimination. Complete bi-directional exon sequencing produces the most reliable data for every position. Agostini: Life Technologies: Employee. Shi: Life Technologies: Employee. Conradson: Life Technologies: Employee. Dinauer: Life Technologies: Employee.