**2. HLA allele number**

cellular interactions and tissue histocompatibility due to cellular discrimination of "self" and "nonself" that require an essential knowledge of the effects of HLA allele matched and mismatched donors in transplantation medicine [4] and transfusion therapy [5]. While the HLA class I molecules are expressed by all nucleated cells to present processed peptides of intracellular origin to CD8+ cytotoxic T cells and serve as ligands for natural killer cells, the class II molecules are expressed by antigen-presenting cells such as B cells, dendritic cells, or macrophages to present exogenous peptides to CD4+ helper T cells of the immune system [6]. In addition, the classical HLA class I genes, HLA-A, HLA-B, and HLA-C, and the classical HLA class II genes, HLA-DR, HLA-DQ, and HLA-DP are distinguished by their extraordinary polymorphisms, whereas the nonclassical HLA class I genes, HLA-E, HLA-F, and HLA-G, are

82 Next Generation Sequencing - Advances, Applications and Challenges

distinguished by their tissue-specific expression and limited polymorphism [2, 3, 7].

for genetic investigation.

The highly polymorphic HLA genomic region is critically involved in the rejection and graftversus-host disease (GVHD) of hematopoietic stem cell transplants [8, 9], the pathogenesis of numerous autoimmune diseases [10–13], and infectious diseases [14]. Apart from regulating immunity, the MHC genes may have a role in reproduction and social behavior, such as pregnancy maintenance, mate selection, and kin recognition [15]. The MHC genomic region also appears to influence drug adverse reactions [16, 17], CNS development and plasticity [18– 22], neurological cell interactions [23, 24], synaptic function and behavior [25, 26], cerebral hemispheric specialization [27], and neurological and psychiatric disorders [28–32]. Hence, the MHC is one of the most biomedically important genomic regions that warrant special attention

In general, the study of the diversity and polymorphic variation of the MHC genomic region has been focused more on humans than any other species and animal population [1] largely because of the high cost and limited throughput of the first generation Sanger sequencing method [33, 34]. However, this is now changing because the next-generation sequencing (NGS) technologies are becoming the method of choice for lower-cost, high-throughput genotyping of MHC genes that are composed of highly homologous multiple loci such as those found in the macaque primate species [35]. Thus, the NGS technologies are expected to perform precise MHC genotyping in human and model animals that already have a collection of MHC allele references, and to facilitate MHC genotyping of wild animals that as yet have no MHC allele references. In addition, the NGS technologies are likely to replace traditional genotyping methods such as subcloning, Sanger sequencing, and previously developed PCR-based MHC typing methods (PCR-RFLP, PCR-SSP, and so on) in the near future. Recently, many articles concerning the development of NGS technologies for precise MHC genotyping and genotyp‐ ing data of MHC genes using the new NGS technologies have been published on the investi‐ gations of human and nonhuman MHC polymorphisms in various fields of study such as

In this chapter, we provide a short review of the current HLA polymorphism information and the use of PCR-based NGS for MHC genotyping in human and nonhuman species, particularly for the Filipino crab-eating macaque MHC (*Mafa*) class I (Mafa-A, -B, -E, -F, and -I) and class

medical science, evolutionary biology, ecology, and population genetics.

II loci (Mafa-DPA1, -DPB1, -DQA1, -DQB1, and -DRB1).


A total of 13,840 HLA allele sequences, 10,297 in the class I and 3543 in the class II gene re‐ gions, were released by the IMGT/HLA database [7] release 3.22 in October 2015 (Table 1).

**Table 1.** Number and genomic distribution (loci) of HLA alleles

The IMGT/HLA database is a specialist database for HLA sequences. Ten years ago, the allelenumbers were only 2182, but since then, the numbers have increased by 1000 allele sequenceseach year. Of 10,297 HLA class I alleles, 3285, 4077, 2801, 18, 22, and 51 alleles were countedin HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G genes, respectively (Table 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.
