[A strategy to clarify ambiguities during genotyping of functional KIR framework genes by sequencing-based typing among ethnic Hans from southern China].

To categorize ambiguous allelic combinations encountered in genotyping of functional KIR genes by sequencing-based typing of the entire coding sequence and develop an efficient approach to identify such ambiguities.Fourteen KIR functional framework genes from 306 ethnic Chinese individuals were genotyped for the entire encoding sequence. The number of ambiguities was directly counted. Based on the differences within each ambiguous allelic combination, group-specific PCR primers and sequencing primers for amplifying the target allelic sequence were designed. The PCR products were then subjected to sequencing in order to identify the ambiguities.The 14 functional KIR genes were subjected to sequencing-based typing (SBT) for the entire encoding sequence. Six ambiguous allelic combinations were identified. The most common ambiguity 3DL2*(002, 007/010, 015) has accounted for 12.09% of the 306 tested samples. The remaining 5 ambiguities were (2DL5A*001, 2DL5B*006/2DL5A*012, 2DL5B*008), 3DL3*(001, 010/009, 048), 3DL2*(007, 008/016, 027), 3DL3*(00801, 048/01001, 026) and 3DL3*(00802, 048/01002, 026) have accounted for 5.88% (18/306), 3.59% (11/306), 2.29% (7/306), 1.31% (4/306) and 1.31% (4/306) of all samples, respectively. For two ambiguities (2DL5A*001, 2DL5B*006/2DL5A*012, 2DL5B*008) and 3DL2*(007, 008/016, 027) subjected to group-specific PCR and re-sequencing, only one demonstrable genotype was identified, While for in each of other four ambiguities subjected to group-specific PCR and re-sequencing, two different genotypes were identified.An efficient approach by group-specific PCR and sequencing retest has been established to clarify the ambiguities during SBT testing for functional KIR framework genes, which may have a broad application in KIR sequencing-based typing.