*2.5.1. Targeting related ambiguities*

Selecting the most appropriate target regions for PCR amplification within a gene or genomic region during primer design is necessary for reasons of technical and cost efficiency. As a result, some exons and introns have to be excluded for some loci, e.g., exon 1 and most of intron 1 of HLA-DRB1. The ambiguity introduced by partial targeting depends on the selection of the non-characterized regions. This is usually a compromise between precision and through‐ put. By analyzing the reference database, it is sometimes possible to omit exons/introns entirely without introducing ambiguity in the genotyping. However, note that consensus sequences will be still less specific by only covering parts of the gene.

Untranslated regions of Class-I loci are rarely targeted, although numerous alleles are differing from each other in a single base in the UTRs. Prime examples are HLA-A\*02:01:01:01 and HLA-A\*02:01:01:02L, the former having a significantly lower expression. The single T → C difference in the middle of the 5'UTR sequence has to be included into the whole gene consensus to precisely determine these alleles. Another example is HLA-B\*35:01:01:01 and HLA-B\*35:01:01:02 where the differentiating SNP is at the end of the 3'UTR: although both 5′ and 3′ UTR has influence to the gene expression after transcription, these parts are often left out from targeting.

Apart from UTRs, some Class-II loci, notably HLA-DRB1, have introns longer than 5 K base pairs incorporating repeats. For many DR loci the targeting primers are usually not in the UTR region, but skipping both exon 1 and the long intron 1 together with the rest of the gene after exon 4, where the remaining exons 5 and 6 are only 24 and 14 bases long, respectively. This makes space for ambiguities such as HLA-DRB1\*12:01:01 vs. HLA-DRB1\*12:10 that are differing in a single SNP on exon 1.
