*4.1.2. Development of multiplex PCR methods*

amplicon. On the other hand, the short-range system is less effective for genotyping recombi‐ nant alleles that have been generated by recombination events of the HLA genes because it is difficult to avoid the phase ambiguities generated by recombinations. For example, in Figure 2, B\*15:20 has an identical nucleotide sequence with B\*15:01 in exon 2 and B\*35:01 in exon 3, but B\*35:43 has an identical nucleotide sequence with B\*35:01 in exon 2 and B\*15:01 in exon 3. When we genotype a DNA sample that has B\*15:01 or B\*15:20 and B\*35:01 or B\*35:43, ambig‐ uous genotyping can result in assignments such as B\*15:01/20 and B\*35:01/43 that are difficult

The long-range PCR system is a method based on PCR amplification of the entire HLA gene region including the promotor-enhancer region, 5′ untranslated region (UTR), all exons, all introns, and the 3′ UTR or partial gene regions that include polymorphic exons and adjacent introns (Figure 1). Primer sets for long-range systems have already been developed and published for HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 (Table 2). The advantage of long-range PCR is that this system can easily solve phase ambiguity even if recombinant alleles such as those shown in Figure 2 are present in DNA samples. Also, the long-range PCR method is expected to detect new poly‐ morphisms and variations throughout the entire HLA gene region. Therefore, the long-range

to assign correctly and definitively.

**Figure 1.** Outline of NGS-based human MHC typing.

86 Next Generation Sequencing - Advances, Applications and Challenges

Recently, we developed four kinds of multiplex PCR methods based on the long-range system for genotyping nine HLA loci (HLA-A, -B, -C, -DRB1/3/4/5, -DQB1, and -DPB1) [54] (Figure 3).

**Figure 3.** Two different nine loci HLA genotyping procedures at the PCR step.

The multiplex PCR methods contributed greatly to simplifying, accelerating, and reducing costs and the number of reagents for the PCR step that is used to prepare samples and libraries for NGS in the NGS-based HLA genotyping method. The multiplex methods also conserved on the amounts of DNA samples needed to genotype a multiple number of HLA loci. Overall, the multiplex PCR method is a powerful tool for providing precise genotyping data without phase ambiguity, with a strong potential to replace the current routine genotyping methods to find polymorphisms. Commercialized PCR amplification reagents such as NEType (One‐ Lambda) that are based on multiplex PCR methods will be made available in the near future, whereas those based on the one-locus, one-tube PCR methods (left side of Figure 3) such as the TruSight HLA panel (Illumina) and NGSgo (GenDX) are already available in the market place.
