**4. Summary of NGS-based human MHC genotyping methods**

Table 2 shows list of publications on NGS-based human MHC genotyping that includes information for PCR range, targeted HLA locus, NGS platform, and allele assignment method. The MHC genotyping methods in human are basically composed of three steps, PCR, NGS, and allele assignment. We summarize the important points in each of the three steps below. The more detailed information is described in our previous publication [52].

#### **4.1. PCR step**

#### *4.1.1. Long- and short-range PCR*

PCR methods produce amplicons of different sequence lengths depending on the primer design and the type of DNA polymerase used for the PCR. The amplicon sizes are usually classified into two size ranges: the short-range system where the amplicon size is <1 kb and the long-range system where the amplicon size is >1 kb as shown in Figure 1.


10,163 and91 alleles were counted in the classical and nonclassical HLA class I genes, respec‐ tively.Of the 3543 HLA class II alleles, 7, 1825, 99, 54, 876, 42, 587, 7, 13, 12, and 13 alleles were countedin HLA-DRA, HLA-DRB1, HLA-DRB3/4/5, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DMA, HLA-DMB, HLA-DOA, and HLA-DOB genes, respectively (Table 1), with3490 and 45 alleles in the classical and nonclassical HLA class II genes, respectively.

Many variations of the conventional HLA genotyping methods such as incorporating restric‐ tion fragment polymorphisms (RFLP) [36], single strand conformation polymorphism (SSCP) [37], sequence-specific oligonucleotides (SSOs) [38], sequence-specific primers (SSPs) [39], and sequence-based typing (SBT), like the Sanger method [33], have been used for the efficient and rapid HLA matching in transplantation therapy [40–43], research into HLA-related diseases [2, 3], population diversity studies [44–46], and in forensic and paternity testing [47]. The HLA genotyping methods mainly applied today are PCR-SSOP, such as the Luminex commercial methodology [48, 49], and SBT by the Sanger method employing capillary sequencing based on chain–termination reactions [33, 34]. However, both methods often detect more than one pair of unresolved HLA alleles because of chromosomal phase (*cis/trans*) ambiguity [50, 51]. To solve the phase ambiguity problem, new HLA genotyping technologies have been reported and commercialized that combine the PCR amplification of targeted HLA genomic regions with NGS platforms such as the ion PGM system (Life Technologies), GS Junior system (Roche), and the MiSeq system (Illumina) [52}. The PCR/NGS methods are expected to produce genotyping results that detect new and null alleles efficiently without phase ambiguity.

**4. Summary of NGS-based human MHC genotyping methods**

The more detailed information is described in our previous publication [52].

the long-range system where the amplicon size is >1 kb as shown in Figure 1.

**4.1. PCR step**

*4.1.1. Long- and short-range PCR*

Table 2 shows list of publications on NGS-based human MHC genotyping that includes information for PCR range, targeted HLA locus, NGS platform, and allele assignment method. The MHC genotyping methods in human are basically composed of three steps, PCR, NGS, and allele assignment. We summarize the important points in each of the three steps below.

PCR methods produce amplicons of different sequence lengths depending on the primer design and the type of DNA polymerase used for the PCR. The amplicon sizes are usually classified into two size ranges: the short-range system where the amplicon size is <1 kb and

**3. History of HLA genotyping methods**

84 Next Generation Sequencing - Advances, Applications and Challenges

**Table 2.** Publication list of NGS-based MHC genotyping in human. Bold letter shows publications from the author's group

The short-range PCR system is a method based on PCR amplification of each exon that includes polymorphic exons 2 and 3 in HLA-A, HLA-B, and HLA-C and exon 2 in HLA-DR, HLA-DQ, and HLA-DP. One of the advantages of the short-range system is that it is the most suitable for application of physically fragmented DNA samples as templates such as those extracted from swabs because the PCR length is relatively short, ranging from 250 bp to 900 bp, per

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

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 to assign correctly and definitively.

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 MHC Genotyping in Human and Nonhuman Species by PCR-based Next-Generation Sequencing http://dx.doi.org/10.5772/61842 87

**Figure 2.** Example of recombinant HLA alleles. B\*15:01 and B\*15:20 and B\*15:01 and B\*35:43 have identical nucleotide sequences in exon 2 and in exon 3, respectively (red boxes), and B\*35:01 and B\*35:43 and B\*35:01 and B\*15:20 have identical nucleotide sequences in exon 2 and in exon 3, respectively (blue boxes). "X" indicates the recombination site.

system is an important and useful alternative to the short-range system for donor-recipient matching in bone marrow transplantation and HLA-related disease studies. In fact, one of the main themes of the upcoming 17th International HLA and Immunogenetics Workshop (IHIWS) in 2017 [53] is "NGS of full length HLA genes," with the following objectives: (1) to complete the sequence of all HLA alleles of the reference cell lines from the 13th IHIWS and (2) to perform HLA genotyping of 10,000 quartet families of varied ancestry, utilizing at least one NGS method.
