Identifying Major Histocompatibility Complex (MHC) Alleles in Rhesus Macaques Using the Next Generation Sequencing MiSeq Platform and an Open Source Allele Identification Program.

Next generation sequencing (NGS) technology has revolutionized the ability to identify important gene sets, including Major Histocompatibility Complex (MHC) alleles, the most polymorphic set of genes in the human and non-human primate genomes. The characterization of MHC class I and II alleles serves various roles in the development of resources and tools needed for vaccine development. In this study, we explored the use of the Illumina MiSeq sequencing system along with the development of software tools to identify MHC class I A and B loci in rhesus macaques. The MiSeq platform combines shotgun sequencing and bridge PCR to provide a high degree of sequence coverage through millions of short reads. The large quantity of reads allows for the statistical exclusion of sequencing errors present in the sequence data. As an alternative to commercially available packages, this study presents the software code for an easily adaptable, user-friendly analysis tool that can be used to genotype rhesus macaques MHC alleles with next generation sequencing data. This tool, referred to as the “Allele Identification Program” (AIP), can be employed by anyone possessing basic computer programming skills. Using the AIP to analyze the MiSeq data proved effective, efficient and inexpensive, potentially making full-length MHC sequencing more accessible for nonhuman primate research. Background & Significance: Animal models play a fundamental role in the strategy of developing vaccines and therapeutics. In this context, instituting accurate and quantitative assessment of specific immune responses is a key objective. This allows for the establishment of animal models that will enhance our understanding of disease pathogenesis. Specifically, the information gained from immunogenetics provides insight into disease prognosis and may also help with interventions. One animal species of extreme importance in the context of animal modeling of human infectious diseases (such as HIV & TB) is the rhesus macaque (Macaca mulatta) (Abee, 2012; Ambrose, KewalRamani, Bieniasz, & Hatziioannou, 2007; Campillo-Gimenez et al., 2010; Chen et al., 1992; Daniel et al., 1985; Gardner & Luciw, 2008; Giraldo-Vela et al., 2008; Harouse, Gettie, Tan, Blanchard, & Cheng-Mayer, 1999; Hatziioannou et al., 2009; Hu et al., 2005; Joag, 2000; Joag, Stephens, Adams, Foresman, & Narayan, 1994; Kanthaswamy, Capitanio, et al., 2009; Kanthaswamy, Gill, et al., 2009; Lackner & Veazey, 2007; Ling et al., 2002; C. J. Miller et al., 1989; Morgan et al., 2008; Reimann et al., 1996; Shedlock, Silvestri, & Weiner, 2009; Uberla et al., 1995; Wambua et al., 2011; Xia et al., 2010). The rhesus macaque is considered to be an animal model of interest for modeling human infectious diseases due to species similarity in disease sequelae (e.g. clinical signs, transmission, susceptibility, and immune response) for several pathogens (Ambrose et al., 2007; Daniel et al., 1985; Harouse et al., 1999; Hatziioannou et al., 2009; Joag, 2000; Lackner et al., 1988; Lackner & Veazey, 2007; Morgan et al., 2008; Reimann et al., 1996; Uberla et al., 1995). Rhesus macaques are also readily available, relatively inexpensive, and their use presents minimal logistical, regulatory, and ethical obstacles (Ambrose et al., 2007; Joag, 2000). In recent years, we and others have characterized the molecular determinants of the rhesus macaque’s cellular immune responses (i.e., Major Histocompatibility Complex (MHC) molecules, motifs, and epitopes) in Indian-origin (Allen et al., 2001; Chen et al., 1992; Dzuris et al., 2000; Giraldo-Vela et al., 2008; Kuroda et al., 1998; Letvin, Miller, Shen, Chen, & Yasutomi, 1993; Loffredo et al., 2008; Loffredo et al., 2007; Loffredo et al., 2005; Loffredo et al., 2004; M. D. Miller, Yamamoto, Hughes, Watkins, & Letvin, 1991; Mothe, Horton, et al., 2002; Mothe, Sidney, et al., 2002; Pal et al., 2002; Reed et al., 2011; Sette et al., 2005; Sidney et al., 2000) as well as the Chinese-origin rhesus macaques for their MHC composition (Karl et al., 2008; A. Li et al., 2012; Loffredo et al., 2005; Loffredo et al., 2004; Maness et al., 2011; Mothe et al., 2013; Mothe et al., 2003; O'Connor et al., 2003; D. Ouyang et al., 2008; D. Y. Ouyang, Xu, Shi, Zheng, & He, 2009; Solomon et al., 2010; Southwood et al., 2011; Wambua et al., 2011; Wiseman et al., 2009). Recent studies have shown that there are appreciable differences between Indian and Chinese’s MHC genes in degrees of polymorphism, specific allelic variants, and functional characteristics (A. Li et al., 2012; Maness et al., 2011; D. Ouyang et al., 2008; D. Y. Ouyang et al., 2009; Solomon et al., 2010; Southwood et al., 2011; Wambua et al., 2011; Wiseman et al., 2009). This is particularly relevant in terms of MHC class I molecules, which encodes a large number of gene products that play important roles in adaptive CD8+ cytotoxic Tlymphocyte cellular immune responses to infections (Bontrop, 2006; Galan, Guivier, Caraux, Charbonnel, & Cosson, 2010; Hurt et al., 2004; Kelley, Walter, & Trowsdale, 2005; Kulski, Shiina, Anzai, Kohara, & Inoko, 2002; Parham, 2005). MHC class I molecules are expressed on all nucleated cells, and its function is to display short peptides derived from intracellular sources on the cell surface for surveillance by CD8+ T-cells (Bjorkman et al., 1987; Bontrop, 2006; Neefjes, Jongsma, Paul, & Bakke, 2011; Parham, 2005; Parham & Ohta, 1996; Peaper & Cresswell, 2008; Williams, Peh, & Elliott, 2002). To ensure a large immunological coverage across an endless amount of potential pathogens, this binding domain is extremely polymorphic and differs between individuals (Abee, 2012; Doxiadis, Otting, de Groot, Noort, & Bontrop, 2000; Dudley et al., 2014; Otting et al., 2007; Parham & Ohta, 1996; Wiseman et al., 2013). The MHC class I molecule is an extracellular heterodimer protein structure composed of three domains (a1-a3) and a B-microglobulin molecule (Bjorkman et al., 1987; Sidney et al., 2008; Sin, Dugdale, Newman, Macdonald, & Burke, 2012). The α1 and α2 domains make up the peptide-binding groove, which will vary in its efficacy to present peptide fragments to cytotoxic CD8+ T-cells (Goulder & Watkins, 2008; Karl et al., 2008; Schatz et al., 2008; Sidney et al., 2008; Solomon et al., 2010). The specific nucleotide sequence which encodes this highly polymorphoric region can heavily influence the outcomes to infections such as human immunodeficiency virus (HIV) (Goulder & Watkins, 2008; A. Li et al., 2012; Maness et al.,