Introduction

Macaque species, such as cynomolgus (Macaca fascicularis, Mafa) and rhesus macaques (Macaca mulatta, Mamu), show close phylogenetic proximity to humans, and they share a common ancestor with humans from approximately 25–33 million years ago [1]. Humans and macaques have a highly related immune system; therefore, macaques are frequently used as non-human primate models for preclinical testing [2]. The use of cynomolgus macaques in biomedical studies has increased due to the limited supply of Indian rhesus macaques after the export ban in 1978 [3]. Cynomolgus macaques are used to study various human diseases such as acquired immunodeficiency syndrome (AIDS) [4], tuberculosis [5], and coronavirus disease 2019 (COVID-19) [6], as well as transplantation [7] and vaccine development [8].

The major histocompatibility complex (MHC) in humans, referred to as the human leukocyte antigen (HLA), plays a crucial role in the innate and adaptive immune responses. HLA is divided into class I, II, and III regions. A single copy of HLA-A, HLA-B and HLA-C is present in the HLA region. The equivalents of HLA-A and HLA-B have been detected in macaques and are named Mamu-A/Mafa-A, Mamu-B/Mafa-B. To date, no orthologs of HLA-C have been identified in macaques. In macaques, the function of HLA-G has been replaced by MHC-AG [9]. The genomic organization of HLA and macaque MHC regions is comparable [10]. Although the number of classical class I A and B genes is increased in macaques due to multiple rounds of duplication [10]. In a rhesus macaque, the presence and physical order of two Mamu-A and nineteen Mamu-B loci have been recorded [11]. Transcriptome studies have shown that the content of Mamu-A and Mamu-B genes vary considerably within each haplotype, leading to different haplotype configurations [12,13,14]. A systematic nomenclature has been established for MHC-A genes [15, 16], and different A genes have been designated A1 to A8 [17, 18]. On a haplotype, a difference in A gene content and combinations has been demonstrated [18,19,20,21]. A haplotype can contain one or two Mamu-A genes with high transcription levels and up to five Mamu-A genes with low transcription levels [13, 14, 19]. More complex gene content and variable transcript levels have been documented for the MHC-B region of macaques [13, 14, 20, 21]. The Mamu-B haplotype contains one to six major transcribed and one to ten minor transcribed Mamu-B genes [13, 14]. As a result, it is not yet possible to assign different B alleles to a particular B gene on a haplotype, and therefore B alleles are named according to the order in which they are discovered on the chromosome [22]. In addition, specific MHC-B alleles have been associated with the progression of several diseases. In humans, HLA-B*35, HLA-B*58, HLA-B*27 and HLA-B*57 showed robust correlations with HIV. For example, HLA-B*35 and HLA-B*58 are associated with rapid disease progression [23]. HLA-B*57 and HLA-B*27 are associated with slower disease progression and lower viral loads [24]. In rhesus macaques, Mamu-B*008 and Mamu-B*017 are known as protective alleles; individuals carrying Mamu-B*08 or Mamu-B*017 exhibited lower viral load and slower disease progression after SIVmac251/SIVmac239 challenge [25, 26]. Interestingly, Mamu-B*08/Mamu-B*017 restricted SIV-derived epitopes share a significant overlap with the peptide binding profile of HLA-B*27/HLA-B*57 [27, 28]. Mamu-B*001 is known to be a protective allele for collagen-induced arthritis (CIA) [29]. Additionally, Mamu-B*001 and Mamu-B*017 are distributed at high frequencies [29, 30]. Macaques carrying these high-frequency alleles are helpful in studying immune protection against various diseases.

MHC class I molecules are the predominant ligands of the killer cell immunoglobulin-like receptor (KIR) family and specific MHC-KIR interactions may be associated with health and disease [31, 32]. The genes encoding MHC and KIR are highly polymorphic, reflected by allelic and copy number variation. KIRs are expressed on natural killer (NK) cells and a subset of T cells [33, 34] and may interact with MHC class I molecules to transduce either an inhibitory or activating signal [35]. KIR genes are located on the leukocyte receptor complex (LRC) on chromosome 19 [36]. The human KIR region has been thoroughly characterized [37, 38] and consists of 17 genes, including 15 expressed genes and 2 pseudogenes [39]. Four genes (KIR3DL3, KIR3DP1, KIR2DL4, and KIR3DL2) are present in all human KIR haplotypes and are referred to as “framework genes” [40]. In humans, KIR haplotypes are divided into two categories, namely group A and group B haplotypes. Group A haplotypes contain a fixed set of seven KIR genes, whereas group B haplotypes contain a more significant variability in the number of genes [41]. Significant similarities have been observed between macaques and their human counterparts [42]. The macaque KIR3DL20 has been detected in all haplotypes and is considered to originate from a common progenitor gene KIR3DL3 in humans [43]. The ortholog of human KIR2DL4 has been termed KIR2DL04 in macaques [44]. A significant difference between human and macaque KIRs is that the KIR lineage II family in macaques has undergone intensive duplication, whereas the expansion of KIR in humans mainly involves lineage III [45]. The KIR region has been studied in rhesus macaques at the genomic [46, 47] and transcriptomic [42, 48, 49]. Multiple studies have sequenced KIR complementary cDNA sequences and used segregation analysis to detect the gene content of each KIR haplotype, showing that different individuals and rhesus macaques of different populations possess diverse KIR gene content [42, 48,49,50,51,52]. The number of KIR genes expressed per animal varies from 4 to 17 in rhesus macaques and 3–13 in cynomolgus macaques [42, 48, 49]. Multiple mechanisms have been shown to drive high variability in the KIR gene system, as evidenced by chromosomal recombination, point mutations, alternative splicing, and stochastic expression [53,54,55]. Given the complexity of KIR genes, long-read sequencing methods are required to improve the quality and continuity of genome assemblies. The combination of Cas9 enrichment and Oxford Nanopore Technologies (ONT) sequencing methods achieved allele-level resolution, which allowed the phasing of six KIR haplotypes in three rhesus macaques [47]. At present, cynomolgus macaque KIR has only been thoroughly studied at the transcriptomic level [48, 56]. However, these transcriptome studies show an unparalleled rapid evolution of the KIR gene region in macaques.

The first human genomic HLA region was successfully sequenced and fully annotated in 1999 [57], whereas a 5.1 Mb genome sequence of the rhesus monkey MHC was constructed and published in 2004 [11]. In a cynomolgus macaque, a BAC contig containing the MHC region was sequenced using a short-read sequencing approach in 2007 [58]. However, the short reads and MHC class I gene duplications resulted in the poor characterization of this region in cynomolgus macaques. Previously, we sequenced the genomes of a cynomolgus macaque and a Chinese rhesus macaque using a whole-genome shotgun strategy on the Illumina HiSeq (2000) platform [59]. However, the quality of the MHC genome assembly was poor because of the limitations of the sequencing technology. Currently, genome assemblies benefit from third-generation sequencing platforms with high accuracy and long read length [60]. For instance, the human MHC region has been characterized in over 20,000 individuals of Han Chinese ancestry using deep sequencing [61]. One study sequenced the KIR region using single-molecule real-time sequencing (SMRT) and phased 16 human KIR haplotypes [37]. Another study designed 18 probes to capture the KIR region of 16 samples and successfully assembled human diploid KIR haplotypes using long-read sequencing. The assemblies covered 97% of the reference genome with 99.97% sequence identity [38]. A high-quality Chinese rhesus macaque reference genome (rheMacs) was built by combining long-read sequencing and multi-platform scaffolding approaches [62]. A new version of the Indian rhesus monkey reference genome (Mmul_10) was assembled using SMRT sequencing, with 66-fold sequencing coverage and 120-fold increase in sequence continuity, as well as high-resolution annotations of MHC and KIR regions [63]. Jayakumar et al. assembled a high-fidelity chromosome-scale cynomolgus monkey genome that was superior in continuity and accuracy [64]. The human HG002/NA24385 genome, which was characterized by highly accurate circular consensus sequencing (CCS) of long reads, performed better in assembly quality and genetic variant detection [65]. The continuous development of third-generation long-read sequencing technologies advances the characterization of complete MHC and KIR gene regions.

This study aimed to assemble complete MHC and KIR genomic regions of a cynomolgus macaque using third-generation long-read sequencing technology.

Materials and methods

Animals and cells

For long-read sequencing, whole blood was collected from an adult Vietnamese cynomolgus macaque (male). In addition, for population analysis of Mafa-B alleles, peripheral blood samples were collected from 33 unrelated and healthy cynomolgus macaques of Vietnamese origin, which were housed in the South China Primate Research & Development Center (Guangdong, China), and peripheral blood mononuclear cells (PBMCs) were isolated.

Pacbio HiFi library construction and sequencing

We extracted 30 μg of high-quality genomic DNA from white blood cells of the male cynomolgus macaque using blood and cell culture DNA kits (QIAGEN). Double-stranded DNA was fragmented, and the size distribution of the sheared DNA was characterized using the DNA 12,000 kit on the Agilent 2100 BioAnalyzer System. DNA fractions of approximately 15 kb were size selected for sequencing. PacBio-CCS sequencing libraries were prepared using the SMRTbell Template Prep Kit v.1.0 (Pacific. No. 100-259-100), according to the manufacturer’s protocol. Four SMRT flow cells were run on the PacBio Sequel II System with the Sequel Sequencing Kit 3.0 chemistry (Pacific Biosciences Ref. No.101-500-400 and 101-427-800) at BGI-Qingdao.

Genome assembly

We used the Unanimity CCS software with the default parameter (–min-passes 3) to process the raw data into HiFi reads (https://github.com/pacificbiosciences/unanimity). Hifiasm (v0.12; -r1- × 0.9-y0.2) and Wtdbg2 (v2.3; -p23-E2S4-s0.05-L5000-X50; -j1500) software tools were used for de novo assembly of the generated HiFi reads [66, 67]. Hifiasm assembly was used to perform an all-versus-all pairwise alignment. Subsequent error correction was applied to remove most sequencing errors. Hifiasm tends to retain as much genome sequence information as possible, especially when dealing with complex regions; therefore, heterozygous variation information is retained [66]. Error-corrected reads were used to generate a draft genome. In addition, we assembled two independent haplotypes to phase complete MHC and KIR haplotypes by processing HiFi reads with hifiasm (v0.12; -r1- × 0.9-y0.2) in the same Vietnamese cynomolgus macaque [66]. The genome assembled by wtdbg2 was generated by directly assembling the raw data and then generating consensus reads through intermediate assembly outputs without eliminating sequencing errors [67]. After considering the results of the hifiasm and wtdbg2 assembly tools, the hifiasm-assembled genome was selected for subsequent genome annotation because the hifiasm assembly was more complete than the wtdbg2 assembly (3.65 Gb; Table 1) and more favorable for assembling complete MHC and KIR regions.

Table 1 Statistics of cynomolgus macaque genome assemblies
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Capture of MHC and KIR genomes

The contiguous MHC region was constructed in three steps. First, coding sequences (CDS) of the MHC genes (humans, rhesus and cynomolgus macaques) were downloaded from the Immuno Polymorphism Database (IPD)-NHMHC (version 3.4.0.0) and IPD-HLA (version 3.39) databases and aligned to the assembled Hifiasm genome using BLAST (v2.2.26) with an alignment length threshold of 500 bp. Second, we used BLAST (v2.2.26) to perform a collinear comparison of the human MHC sequences (chromosome 6:28,510,021–33,480,578) and the candidate MHC contigs obtained above. Third, the MHC CDS of cynomolgus monkeys were collinear compared with contig utg000348l (which represents the assembled MHC cluster) using BLAST (v2.2.26). The collinearity block length threshold was set to 200 bp. For KIR region analysis, CDS of KIR genes (humans, rhesus and cynomolgus macaques) from the IPD-NHKIR (version1.3.0.0) and IPD-KIR (version 2.10.0) databases were aligned to the assembled genome using BLAST (v2.2.26). The alignment length threshold was set at 100 bp.

Gene annotations

Gene annotations included four aspects: repetitive sequence annotation, gene structure annotation, gene function annotation, and non-coding RNA (ncRNA) annotation (Additional file 1: Fig. S1). Two methods were used for repetitive sequence annotation: homology-based and de novo. We used RepeatMasker (v4.0.6) software (http://repeatmasker.org/) for homologous annotation based on Repbase (release 21.01) (http://www.girinst.org/repbase). Based on the sequence alignment of the genome itself, we used RepeatModeler (v2.0.1) [68], Piler (v1.0) [69] and RepeatScout (v1.0.6) [70] for gene annotations. Based on the characteristics of the repeat sequence, we used TRF (v4.07b) [71] and LTR-FINDER (v1.0.7) [72] for de novo annotation. Three sources of gene structure annotation were used: homolog annotation, de novo prediction, and transcript annotation. For homolog annotation, we selected protein sequences of six different species (Homo sapiens, Macaca fascicularis, Monodelphis domestica, Mus musculus, Otolemur garnettii, and Pan troglodytes) and compared them with the assembled genome using the software tool Genewise (v2.4.1) [73]. De novo prediction was performed using the software tools Augustus (v3.2.3) [74] and Genscan (v1.0) [75]. We then used a combination of the software tools Pasa (v.2.0.2) + Transdecoder (v.3.0.1) [76, 77] for transcript annotation. Finally, EVM (v1.1.1) [78] software was used to integrate the above-mentioned evidence sets and to filter out genes based on the following conditions: a gene has only one type of evidence supported by de novo prediction, the CDS length is short (< = 150 bp), and the overlap length ratio with TE is less than 0.2. The completeness of gene structure annotations was evaluated using BUSCO (v3.0.2), utilizing the Vertebrata odb9 set of 2,586 genes. Protein sequences obtained by gene structure annotation were compared with protein databases (SwissProt/TrEMBL (Release 2020_03, June 17, 2020) [79], KEGG (Release 94.2, June 1, 2020) [80], and InterPro (Release 80.0, June 18, 2020) [81] for functional annotation. For ncRNA annotations, we used tRNAscan-SE (v3.0) [82] software to search for tRNA sequences in the genome. Since rRNA is highly conserved, we used the rRNA sequences in the Rfam (v12.0) database as reference sequences to search for rRNA in the assembled genome by comparison with the RNAmmer (v1.2) tools [83]. In addition, the Rfam (v12.0) database and Infernal (v1.1) software [84] were used to predict the miRNA and snRNA sequences in the assembled genome.

We annotated the MHC genes using the previously annotated human MHC (NC_000006.12, BA000025.2), rhesus macaque MHC (NC_041757.1, AC148659-AC148717, AB128049.1), and cynomolgus macaque MHC (NC_022275.1) regions. Manual annotation was uniformly performed on the sequences. We used Blastn (v2.12.0, NCBI) to confirm the documented genes within the genomic sequences. Confirmed genes from this newly assembled genome were identical to previously assembled cynomolgus macaque cDNA sequences from the database or were orthologs to documented human or rhesus macaque genes. ncRNA and small nucleolar RNA (snoRNA) sequences were annotated based on human MHC sequences, whereas pseudogenes were defined as nonfunctional copies of reported genes with their coding regions disrupted by premature stopcodons and/or frameshift mutations. We annotated the KIR genes using exon sequences of rhesus and cynomolgus macaques from the IPD-NHKIR database (Release 1.3.0.0). The confirmed Mafa-A/AG/B and Mafa-KIR gene sequences were submitted to IPD to receive official designation [85, 86].

RNA extraction, cDNA cloning, and sequencing

We used E.Z.N.A.™ Blood RNA Kits (OMEGA Bio-tek) to extract total RNA from PBMC samples of 33 unrelated and healthy cynomolgus macaques of Vietnamese origin. cDNA was synthesized using the PrimeScriptTM II 1st Strand cDNA Synthesis Kit (TaKaRa Bio, Kusatsu, Japan). For the specific amplification of ten Mafa-B loci, locus-specific primer sets were used to amplify exons 2 and 3. The forward primers were located in exons 1 or 2, and the reverse primers were located in exons 3, 4, or 5. The polymerase chain reaction (PCR) cycle conditions consisted of a denaturation process for 5 min at 95 °C, followed by 34 cycles at 95 °C for 30 s, 58 °C to 64 °C for 30 s, 72 °C for 25–50 s, and a final step at 72 °C for 10 min (Additional file 1: Table S1). PCR was performed in a 50 µL reaction mixture using Green Taq DNA mix (Vazyme). The PCR products were purified and ligated to the pMD19-T vector (TaKaRa). Ligations were transformed into Escherichia coli DH5α competent cells. Approximately 10–50 clones were selected for each Mafa-B amplicon from each animal and sequenced on an automatic DNA sequencer (ABI3730XL) by a service provider (Tsingke, Guangzhou, China). Nucleotide sequences of cDNAs were analyzed using SeqMan (DNASTAR, Madison, WI, USA) [87] and aligned using SnapGene 4.1.9(GLS Biotech, https://www.snapgene.com) and the Clustal W program (BioEdit) [88]. When a sequence was identical in at least three clones, it was considered an allele. These sequences were then submitted to GenBank for accession numbers.

Results

De novo assembly of the cynomolgus macaque genome

A total of 98.2 Gb of HiFi reads were obtained, with an average subread length of approximately 14 kb. Long-read assemblies (30 ×) of this cynomolgus macaque genome were generated using Hifiasm and Wtdb2 and yielded total sizes of 3.7 Gb and 2.7 Gb, respectively. The N50 sizes of contigs reached 12.1 Mb and 13.7 Mb, respectively; and the overall GC contents of the two assemblies were 41.16% and 40.99% (Table 1). Gene structures were annotated using EVM and predicted 31,606 genes, of which 81.41% were considered to be functional (Additional file 1: Tables S2, S3 and Fig. S2). BUSCO evaluation showed that 91.60% of the complete genes were fully annotated (Additional file 1: Table S4). In addition, ncRNA sequences were annotated in the cynomolgus macaque genome (Additional file 1: Table S5). This genome contained 49.23% repeat sequences that could be classified into different subtype elements, of which the majority represented long interspersed nuclear elements (LINEs) (Additional file 1: Fig. S3 and Tables S6, S7). In addition, two independent haplotypes of this cynomolgus macaque genome assembled with Hifiasm yielded total sizes of 3.1 Gb and 2.9 Gb, respectively, with N50 contigs of 16.9 Mb and 15.0 Mb (Additional file 1: Table S8).

Physical mapping of the cynomolgus macaque MHC cluster

Contig utg000348l (total length 8,094,345 bp) displayed collinearity with the humans (MHC region on chromosome 6) and cynomolgus macaque MHC sequences from the IPD database (Fig. 1a, b). It was defined as harboring the complete MHC region, including all class I and II genes (Fig. 1a, b).

Fig. 1
figure 1

Collinearity between cynomolgus macaque contigs (utg000348l and utg000460l) and published MHC and KIR sequences, respectively. a BLAST (v2.2.26) was used to perform a collinear comparison of the human MHC sequences (chr6: 28,510,021–33,480,578) and the candidate MHC contig utg000348l. The left side of the figure represents the human MHC sequence (green), and the right side of the figure represents the sequence of the candidate MHC contig utg000348l (purple). b The MHC CDS sequences of cynomolgus macaques downloaded from the IPD were collinearly compared with the candidate MHC contig utg000348l using BLAST (v2.2.26). The top of the figure represents the MHC CDS sequences of cynomolgus macaques downloaded from the IPD (orange), and the bottom of the figure represents the sequences of the candidate MHC contig utg000348l (purple). c The KIR CDS sequences of human downloaded from the IPD were collinearly compared with the candidate KIR contig utg000460l using BLAST (v2.2.26). The top of the figure represents the KIR CDS sequences of human downloaded from the IPD (pink), and the bottom of the figure represents the sequences of the candidate KIR contig utg000460l (blue). d The KIR CDS sequences of cynomolgus macaques downloaded from the IPD were collinearly compared with the candidate KIR contig utg000460l using BLAST (v2.2.26). The top of the figure represents the KIR CDS sequences of cynomolgus macaques downloaded from the IPD (green), and the bottom of the figure represents the sequences of the candidate KIR contig utg000460l (blue)

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This cynomolgus macaque MHC region in contig utg000348l spans 5.08 Mb, which is similar to the 5.1 Mb MHC region defined on the reference genome of rhesus macaques, considering the same start and end positions (Fig. 2) [11]. This contiguous region contained 453 genes that were annotated from GABBR1(located telomeric to the extended class I region) to KIFC1 (located at the end of the extended class II region). Of these genes, 169 were predicted to be functional, 53 were classified as ncRNA, 5 genes were classified as snoRNAs, and the remaining 226 genes were classified as pseudogenes (Additional file 2: Table S9). Overall, a high level of conserved synteny was observed for cynomolgus and rhesus macaque MHC clusters with respect to functional genes as well as many pseudogenes. This assembled cynomolgus macaque MHC region shows an extension in size when compared to the HLA region of the human reference genome (hg38). This radical difference in size is the result of significant expansions within the macaque MHC-A and -B regions. The overall gene content in class II and III regions of this cynomolgus macaques overlaps with that of humans to a large extent. Five protein-coding genes (HLA-C, BTNL2, HLA-DQA2, HLA-DQB2, and HLA-DRB5) that were found in the HLA region had no orthologs in this cynomolgus macaque. Identical genes were defined in the MHC clusters of the two macaque species, except for the SMIM40 gene, which was only identified in this cynomolgus macaque.

Fig. 2
figure 2

Linear representation of the cynomolgus and rhesus macaque MHC. The position and name of each gene in the MHC region are indicated above or below the horizontal line according to the convention, and in concordance with the forward or reverse orientation of a gene, respectively. The gene content of this cynomolgus macaque MHC in contig utg000348l was established by comparison with the rhesus macaque MHC (Daza-Vamenta et al., 2004; Jerzy K Kulski et al., 2004). For the cynomolgus and rhesus macaque MHC region, the positions of the pseudogenes are indicated on a separate horizontal line. Between both the cynomolgus and the rhesus macaque, the class I region ranges from MHC-F to MIC2 (blue), the class III region ranges from PPIAP9 to RNU6-603P (green), and the class II region ranges from MHC-DRA to MHC-DPA3 (pink). There are some genes followed by a number (such as “−1”, “−2” and “−3”) to distinguish between different copies of the gene. The cynomolgus genes (top) and the rhesus genes (bottom) are scaled in kilobase pairs

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Characteristics of the cynomolgus macaque MHC class I region

The composition and organization of the MHC region in contig utg000348l are considerably parallel between humans and macaques to a great extent. An exception was found for class I genes. In macaques, remodeling by 'birth and death' evolution resulted in an expansion of the number of class I genes, most likely in response to environmental pathogens. Even though a substantial fraction of MHC class I genes feature high conservation and homology, the number of genes with classical and non-classical characteristics within the MHC class I region differs extensively between humans and the two macaque species.

The Mafa-A region was subjected to duplication events, with three Mafa-A genes located in this assembled MHC region (Figs. 2, 3a). Five copies of Mafa-AG genes were identified as functional genes in this assembled MHC region (Figs. 2, 3a). Two copies of Mafa-E located close to each other were detected (Figs. 2, 3a). Six pseudogenes (Mafa-59, Mafa-70, Mafa-92, Mafa-75, Mafa-80, and Mafa-30) were identified as orthologs of human pseudogenes (Additional file 2: Table S9).

Fig. 3
figure 3

Comparative genomic map of the MHC and KIR region in human, rhesus and cynomolgus macaque. a Comparative genomic map of the protein-coding MHC genes in human (HLA), rhesus macaque (Mamu) and cynomolgus macaque (Mafa). Red and blue boxes indicate MHC class I and class II genes, respectively. Distances between genes are not scaled. b Comparative genomic map of the HLA-B, Mamu-B and Mafa-B regions (contig utg000348l). Distances between genes are not scaled. The table on the right shows the Mafa-B alleles with the highest frequency in our study (frequencies in between brackets). c Comparative genomic map of the KIR genes in human (haplotype A and B), rhesus and cynomolgus macaque (contig utg000460l). Distances between genes are not scaled. Purple represents activating KIR genes, green represents inhibitory KIR genes, pink represents pseudogenes, and KIRID genes are shown in yellow. Due to the high diversity of KIR genes in rhesus macaques, and the variable copy number of KIR genes per haplotype, the specific names of the KIR genes in rhesus macaques are not indicated in the figure

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This assembled MHC region containing ten Mafa-B genes spans approximately 500 kb in contig utg000348l (Figs. 2, 3b). Of the ten Mafa-B genes, one has a stopcodon within exon 1 that presumably inactivates the gene. In comparison, 19 different Mamu-B genes were defined in the reference rhesus macaque MHC genome, 14 of which may encode proteins [11]. In addition, we detected a Mafa-I gene in this assembled MHC region (Fig. 2 and Additional file 2: Table S9). The I gene is the result of a duplication of the B gene and is also present in rhesus macaques [89].

In macaques, exon 1 of the MHC-B genes can contain one or two start codons. In this assembled MHC region, we observed that Mafa-B1, Mafa-B6, and Mafa-B8 had two start codons, of which the first was out-of-frame and most likely the second was used as the start codon. Three other genes, Mafa-B2, Mafa-B3, and Mafa-B5, had two in-frame start codons, with the second ATG located at amino acid position 4 in the coding region (ATGCGGGTCATG). Only one start codon was identified in Mafa-B4, Mafa-B7, Mafa-B9, and Mafa-B10.

Overall, the majority of the genes present in this MHC region are conserved in humans, rhesus macaques, and cynomolgus macaques. Differential selective conditions, such as pathogen encounters, might have driven this extensive expansion observed in the macaque MHC class I region, which was not observed in humans.

Transcription status of the ten identified Mafa-B genes in a panel of Vietnamese cynomolgus macaques

To elucidate which of the ten B genes identified on this Vietnamese cynomolgus MHC haplotype in contig utg000348l (Figs. 2, 3b) may encode a functional molecule and to identify high-frequency alleles, we performed Mafa-B locus-specific amplification in a cohort of 33 Vietnamese cynomolgus macaques (Additional file 2: Table S10). A total of 6,437 clones were sequenced, and 5,351 Mafa-B cDNA sequences were acquired. In the panel of 33 animals, we have identified 92 Mafa-B sequences (OK486180-OK486272). Most of these sequences contain only partial exons 2 and 3. Sixty-five of the sequences may represent novel alleles, and 27 of the sequences were identical to those reported previously in the IPD-MHC database [90]. Our results showed that the B2, B3, B5, and B6 genes might be functional genes and are transcribed at abundant levels (Additional file 2: Table S11). Amplification with B2 locus-specific primers resulted in 22 of the 33 cynomolgus macaques detecting a Mafa-I*01-like transcript. For B3 locus-specific amplification, Mafa-B*056 was observed in 14 animals. At the B5 locus, Mafa-B*034 was observed in 11 individuals, and our study showed a distribution frequency of 24.2% (8/33) for Mafa-B*001 at the B6 locus. Furthermore, for the B7 locus, Mafa-B*079 and Mafa-B*017 were detected in 6 and 2 animals, respectively. For B1, B4, and B9 locus-specific amplification, we observed no transcripts (B1 and B4) or alleles with low transcription levels (B9).

Characteristics of the cynomolgus macaque MHC class II regions

The MHC class II regions (MHC-DRA, -DQA1, -DQB1, ­DOB, -DMB, -DMA, -DOA, -DPA1 and -DPB1) are well conserved in humans and macaque species, except for the MHC-DRB, MHC-DQA2, and MHC-DQB2 genes [91]. Five DRB genes were defined in this assembled MHC region of contig utg000348l, accompanied by one DQA gene and one DQB gene (Additional file 2: Table S9). Of these genes, DRB2 and DRB9 were pseudogenes. In rhesus macaques, the MHC class II region is similar to that in this cynomolgus macaques. In humans, the number of DRB genes present per haplotype ranged from one to four, and two DQA genes and two DQB genes could be identified [92]. The order of the genes within the human and macaque MHC class II regions was nearly identical.

Genomic mapping of the cynomolgus macaque KIR gene region

Human and cynomolgus macaque KIR sequences from the IPD-KIR database displayed collinearity with contig utg000460l (total length: 1,114,979 bp) (Fig. 1c, d). This contig comprised the complete KIR haplotype, ranging from 765,301 to 944,995, thereby spanning a total length of 179,694 bp.

This assembled KIR region in contig utg000406l comprises 12 KIR genes, and the sequence similarity of the coding regions reached 98% to 100% compared to the cDNA references. This constructed KIR region was flanked by the LILRA6 and FCAR genes, indicating that one complete KIR haplotype was assembled. All 12 KIR genes were organized in a head-to-tail arrangement and tightly clustered within 179 kb (Fig. 3c and Additional file 2: Table S12). The length of a single KIR gene varies from 8.5 to 15.1 kb. Three KIR sequences have been reported previously, whereas the remaining nine alleles were novel and received official designations. This assembled centromeric region comprises three genes, including the framework gene, Mafa-KIR3DL20, and a pseudogene, Mafa-KIRDP. The Mafa-KIR1D encodes receptors with a single extracellular domain and is thereby distinct from human lineage III KIR [45]. A KIR2DL04 gene was identified in the telomeric region, which is conserved in humans and two macaque species [42]. In addition, eight lineage II KIR genes comprise approximately 115 kb of this assembled telomeric region, five of which are inhibitory genes, whereas three genes encode activating receptors.

Characteristics of the cynomolgus macaque MHC class I region in two independent haplotypes

In haplotype 1, contig hltg000223l (total length 8,100,161 bp) displayed collinearity with MHC contig utg000348l (Additional file 1: Figs. S4, S5). In haplotype 2, three contigs showed collinearity with the MHC contig utg000348l (Additional file 1: Figs. S4, S5). They are as follows: h2tg000276l (total length 1,823,061 bp), h2tg000318l (total length 3,049,176 bp), and h2tg000147l (total length 6,336,003 bp). Of note, MHC class I genes were located on contigs h2tg000318l and h2tg000147l of haplotype 2 (Additional file 1: Fig. S6).

The assembled contig hltg000223l of haplotype 1 comprises five Mafa-AG, three Mafa-A and ten Mafa-B genes (Additional file 1: Fig. S7), ranging from 2,744,163 to 5,409,505 (Additional file 2: Table S13). The cDNA and genomic sequences of the MHC class I genes in contig hltg000223l were 100% identical to their counterparts on previously assembled MHC contig utg000348l, except for the Mafa-A2 and Mafa-B4 genes. The Mafa-A2 gene in MHC contig utg000348l was named Mafa-A1*090:08:01:01 N and had an early stopcodon in exon 3. But the Mafa-A2 gene in haplotype 1 has a deletion of two bases in exon 1, resulting in this Mafa-A2 gene identical to allele Mafa-A1*090:04:02. Compared to the Mafa-B4 gene (Mafa-B*109:30:01:01) in contig utg000348l, the Mafa-B4 gene (Mafa-B*109:17:01:01) in haplotype 1 has one base deletion in exon 7.

Four Mafa-AG, two Mafa-A and sixteen Mafa-B genes were defined in haplotype 2 (Fig. S7), ranging from 892,631 to 3,028,576 in contig h2tg000318l and ranging from 40,057 to 659,990 in contig h2tg000147l (Additional file 2: Table S14). Of which, four Mafa-AG, two Mafa-A and seven Mafa-B genes were identical to the cDNA and genomic sequences of published MHC alleles. The remaining nine Mafa-B genes were novel and received official designations.

Genomic mapping of the cynomolgus macaque KIR gene region in two independent haplotypes

In haplotype 1, contig hltg000304l (total length: 1,118,988 bp) displayed collinearity with KIR contig utg000406l (Additional file 1: Figs. S8 and S9). In haplotype 2, contigs h2tg000218l (total length 4,001,668 bp) and h2tg000293l (total length 633,137 bp) showed collinearity with the KIR contig utg000406l (Additional file 1: Figs. S8 and S9). However, all KIR genes are located on contig h2tg000293l of haplotype 2 (Additional file 1: Fig. S10).

Contig hltg000304l of haplotype 1 contains twelve KIR genes (Additional file 1: Fig. S11), ten of which are identical to the cDNA and genomic sequences of KIR genes on contig utg000406l. Two KIR genes (Mafa-KIR3DSW22*004:02 and Mafa-KIR3DLW14*003:02) have subtle base differences in the intron region with their counterparts located in contig utg000406l (Additional file 2: Table S15). All twelve KIR genes were clustered within 179 kb, ranging from 768,364 to 948,057 in contig hltg000304l of haplotype 1.

Eight KIR genes were defined in haplotype 2 (Additional file 1: Fig. S11), ranging from 339,299 to 464,543 in contig h2tg000293l (Additional file 2: Table S16). The sequence similarity of the coding region is 97% to 100% compared to the cDNA references. Two KIR genes have been reported previously, whereas the remaining six KIR alleles were novel.

Similar to the MHC class I region in macaques, the KIR gene region does not follow the standard organization. The gene content per haplotype displayed extensive diversity, as has been previously demonstrated for MHC and KIR haplotypes in cynomolgus and rhesus macaques [12,13,14,15,16, 41,42,43,44,45,46,47,48].

Discussion

A contiguous and accurate cynomolgus macaque genome was de novo assembled using hifiasm and wtdbg2 with N50 contigs of 12.1 Mb and 13.7 Mb. Long-read sequencing was performed using the PacBio platform to characterize a complete reference cynomolgus macaque genome and reached a 30-fold coverage.

In the past decade, long-read sequencing techniques have developed rapidly, improving the continuity and quality of whole-genome assemblies. The assembled rheMacS increased the overall contiguity by 75-fold, closing 21,940 gaps of the previous assembly rheMac8 [62]. Compared with the rheMacS assembly, the cynomolgus macaque genome we assembled displayed less fragmentation (4741 vs. 2468/1397 contigs; 8.19 Mb vs. 12.14 Mb/13.77 Mb contig N50 length) [62]. The Mmul_10 genome assembly greatly improved the contiguity and completeness of the rhesus macaque reference genome with a contig N50 of 46 Mb [63]. Nonetheless, gaps still exist in the Mamu-B and KIR regions of the Mmul_10 assembly. Long-read data are particularly advantageous in resolving complex genomic regions, especially those with high repetitiveness and abundant GC content. The cynomolgus macaque genome assembled in this study contains two multi-gene families, MHC and KIR clusters, which are located on gap-free contigs. Currently, the representative genome of cynomolgus macaques is the chromosome-level assembly MFA1912RKSv2. The cynomolgus macaque genome we assembled with Hifiasm is 3.7 Gb, larger than the current cynomolgus monkey genome MFA1912RKSv2 (2.8 Gb) [64]. However, the contig N50 of the cynomolgus macaque genome assembled in this study was smaller than that of the MFA1912RKSv2 assembly. Despite this, the precise allelic-level annotation of the complex regions of MHC and KIR gene families with high content variability demonstrated that the genome assembly was accurate.

The MHC and KIR gene families experience various rounds of expansion and contraction facilitated by recombination events [13, 53]. In addition, both immune gene systems feature highly variable gene content and complicated sequence similarity, making the rapid genomic characterization of these multigenic families a challenge. Whereas short-read approaches hamper the complete and accurate characterization of these complex regions, SMRT sequencing platforms enable a comprehensive characterization of the MHC and KIR regions. Our cynomolgus macaque genome assembly was constructed from relatively long and high-accuracy contigs, allowing us to characterize complex regions that display extensive polymorphism and copy number variation. In addition, we phased complete MHC and KIR haplotypes in this cynomolgus macaque genome and comprehensively annotated genes located on the extended MHC class I and KIR region. The cDNA sequences of MHC class I and KIR genes on haplotype 1 are identical to their equivalents on contigs utg000348l and utg000406l, except for Mafa-A2 and Mafa-B4 genes. The two Mafa-A2 genes have two bases difference in exon 1 and the two Mafa-B4 genes have only one base difference in exon 7. Despite these slight base-level differences, but this demonstrates that the assembly strategy we have applied effectively phased diploid haplotypes without additional sequencing data. Together, four Mafa-AG, two Mafa-A, seven Mafa-B and two KIR genes in haplotype 2 were completely identical to previously described sequences of cynomolgus macaques. This further supports that our assemblies are precise at the allele level.

Different numbers of MHC class I genes were identified in this newly assembled cynomolgus macaque genome, it substantiates the diverse genetic content of this complex region. In rhesus macaques, one to six Mamu-A genes and up to nineteen Mamu-B genes can be present in a haplotype. A haplotype usually contains one or two major transcribed and up to five minor transcribed Mamu-A genes [13, 14]. For Mamu-B genes, one to six major transcripts and one to ten minor transcripts can be present per haplotype [13, 14]. This situation is similar in cynomolgus macaques. The number of Mafa-A and Mafa-B genes varies from one to six and one to seventeen per haplotype, respectively [93,94,95,96]. One to two major transcribed and up to five minor transcribed Mafa-A genes may be detected per haplotype [93,94,95,96,97]. A haplotype can comprise one to seven major transcribed and up to fifteen minor transcribed Mafa-B genes [93,94,95,96,97]. Our data illustrated that the number of Mafa-A and Mafa-B genes varies among haplotypes in this assembled cynomolgus macaque genome. The Mafa-B regions contain ten and sixteen Mafa-B genes in haplotype 1 and haplotype 2, respectively. Despite this, it represents only two haplotypes in cynomolgus macaque. One study showed that one detected Mamu-B haplotype matched the Mamu-B region in rhesus MHC published in 2004, with only eight Mamu-B transcripts observed, whereas no transcripts were detected for the other Mamu-B loci [11, 98]. This indicates that most Mamu-B genes are not transcribed. The high variability in gene copy number combined with the differential transcription levels in MHC class I genes highlight a different selective sweep occurring in their MHC class I repertoire in macaques. Although there is an evident discrepancy in the variation of MHC class I genes between the two widely used non-human primate animal models, many studies have demonstrated that a few conserved MHC class I genes in both macaque species may have evolved to fulfill important immune functions. These conserved genes may have fine-tuned their sequences in response to environmental pathogens [14, 93, 99]. Our previous studies have confirmed that ancient introgression occurs at the junction of the two species, as extremely high nucleotide sequence similarity was observed between Chinese rhesus macaques and cynomolgus macaques [59]. A comparison of rhesus and cynomolgus macaques as models of COVID-19 infection showed that both species responded similarly to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection when challenged with SARS-CoV-2 [100]. The high rate of gene overlap and similar immune responses to infection will advance the widely use of cynomolgus macaques as preclinical animal models in biomedical research to study human diseases.

KIR genes are characterized by homology, duplication, and structural diversity in macaques, making this region increasingly complex. Studies of the KIR region in rhesus and cynomolgus macaques showed a differential number of KIR genes among different haplotypes and populations [48,49,50,51,52]. Characterization of the KIR transcriptome of 298 Indian rhesus macaques using SMRT sequencing yielded 112 unique KIR haplotype configurations, and each haplotype contains 4 to 17 different KIR genes [42, 48]. Based on published transcriptome data, a cynomolgus macaque haplotype contains 3 to 13 different KIR genes [48]. Our currently assembled cynomolgus macaque KIR haplotypes represent only two combinations of KIR genes and differ in gene content. Many MHC class I and class II genes have been shared in rhesus and cynomolgus macaques [14, 95, 101]. However, only a few KIR alleles were shared between the two macaque species. The occurrence of chromosomal recombination, point mutations, alternative splicing, and stochastic expression results in a large number of orthologous and species-specific KIR genes [53]. This indicates that different selective forces drive the evolution of the KIR system, as evidenced by differences in lineage expansion and haplotype configurations between rhesus and cynomolgus macaques. Our extended knowledge of the relatively high levels of species-specific KIR genes indicates considerable diversity and complexity, whereas the homologous genes shared between the two highly related macaque species reflect a common ancestry. A comprehensive overview of KIR genes is fundamental for the study of KIRs, which advances the study of using macaques as a model and facilitates future studies on the role of KIRs in immunogenetics. The extensive diversity of MHC class I molecules may have prompted the rapid selection of KIR molecules. This can be readily understood in the context of our understanding of the highly polymorphic MHC class I molecules in macaques, as MHC class I molecules are specific ligands for KIR molecules. Thus, the extreme complexity of macaque KIR molecules illustrates their coevolution with MHC ligands. Different sets of KIR and MHC genes have been associated with the progression of AIDS [102], hepatitis C [103], reproduction [104, 105] and hematopoietic stem cell transplantation [106]. A holistic analysis of MHC and KIR genes may contribute to a better understanding of immunogenetics by studying the complex functions of MHC/KIR pairs.

It was found that Mafa-B genes have the highest degree of duplication among the class I genes. We performed locus-specific amplification of ten Mafa-B loci in 33 cynomolgus macaques of Vietnamese origin. We identified four functional loci, B2, B3, B5, and B6 at the transcriptome level, with Mafa-I*01, Mafa-B*056, Mafa-B*034, and Mafa-B*001 lineages displaying the highest frequencies. For most macaque B haplotypes, two or three genes are transcribed at substantial levels, which are thought to fulfill the classical MHC antigen-presenting function [93,94,95,96,97]. The highly frequent I gene has the characteristics of classical and non-classical genes and most likely executes a more specific function [107]. A previous study found that the peptide Gag QI9 was presented by Mamu-A1*001:01 and also presented by Mamu-B*056:01 [108]. In a rhesus macaque SIV infection model, Mamu-A1*001:01 was identified as a protective allele [109]. It would be interesting to test whether the highly frequent Mafa-B*56 lineage alleles that we identified, which are orthologs of the Mamu-B*56 lineage, can also present Gag QI9. For the B11L gene (at the B6 gene), we identified several transcripts orthologous to B11L with a frequency distribution of 21.2% (7/33). The Mamu-B*001 allele confers resistance to CIA [29]. In our study, we found that the distribution frequency of Mafa-B*001 was 24.2% (8/33) at the B6 locus. However, whether Mafa-B*001 is resistant to the CIA is currently unknown. Of the ten B loci, B7, B8, and B10 genes have been reported to be pseudogenes [10]. However, for the B7 locus, we detected alleles of the Mafa-B*079 and Mafa-B*017 lineages in 6 and 2 animals, respectively. Of these, Mafa-B*017:02 is homologous to Mamu-B*017:01. Mamu-B*017:01 controls SIV replication and disease progression [26]. The B1, B4, and B9 genes were reported to have low transcription levels [93,94,95,96,97], and only three alleles (Mafa-B*180:02:01:02nov, Mafa-B*124:03:01:01nov, Mafa-B*021:07nov) were detected in the B9 locus. The combinations I*01-B*056:05-B*034:04 (animal 1, 5, 6, 18, 21) and I*01-B*034:04-B11L*01 (animal 16) matched the Mafa-B region in contig utg000348l. In addition, the combinations B*105:01-B*156:01 (animal 10) and B*105:01-B*001:01 (animal 33) matched the Mafa-B region in haplotype 2.

However, our sample size was too small to include all Mafa-B alleles. Clone-based strategies are expensive and time-consuming, and amplification may not recover clones sufficiently, resulting in transcripts at low transcription levels that may not be identified. In addition, the sequence similarity between MHC genes makes the design of specific primers difficult, and a large number of primer pairs must be designed to enable comprehensive genotyping. Nevertheless, it may be a relatively effective PCR genotyping method to design primers to amplify amplicons spanning the highly conserved peptide-binding domain of MHC class I molecules [12]. A strategy was developed for KIR genotyping by designing primers based on highly conserved sequences in the D1 domain (most of the region), the D2 domain and the stem region (part of the region) [52]. Comprehensive MHC and KIR genotyping is important for better understanding the role of these polymorphisms in human disease models.

This study has limitations, as the MHC and KIR genomic regions analyzed here represent only one example in cynomolgus macaques. Therefore, additional haplotypes at the genomic level are required to obtain a comprehensive overview of the complexity of these immune regions in this species, as well as supplementary studies to generate the complete MHC and KIR genotypes and compare the detected genes to this currently assembled genome.

Conclusions

We constructed full-length MHC and KIR regions in a Vietnamese cynomolgus macaque. There were 453 loci in the extended MHC region and 12 loci in the KIR cluster in this new reference cynomolgus macaque genome. The Mafa-B genes displayed the highest degree of duplication among MHC class I genes. We identified four functional Mafa-B loci in this cynomolgus macaque MHC region. The gene content of the MHC class I region and KIR region is highly variable between the two independent haplotypes in this assembly. Knowledge gained on the genetic organization of MHC class I and KIR genes in macaques contributes to the understanding of how the immune system evolved and lays the foundation for investigating NK cell responses in non-human primate models.