Major histocompatibility complex haplotyping and long-amplicon allele discovery in cynomolgus macaques from Chinese breeding facilities

Abstract

Very little is currently known about the major histocompatibility complex (MHC) region of cynomolgus macaques (Macaca fascicularis; Mafa) from Chinese breeding centers. We performed comprehensive MHC class I haplotype analysis of 100 cynomolgus macaques from two different centers, with animals from different reported original geographic origins (Vietnamese, Cambodian, and Cambodian/Indonesian mixed-origin). Many of the samples were of known relation to each other (sire, dam, and progeny sets), making it possible to characterize lineage-level haplotypes in these animals. We identified 52 Mafa-A and 74 Mafa-B haplotypes in this cohort, many of which were restricted to specific sample origins. We also characterized full-length MHC class I transcripts using Pacific Biosciences (PacBio) RS II single-molecule real-time (SMRT) sequencing. This technology allows for complete read-through of unfragmented MHC class I transcripts (~1,100 bp in length), so no assembly is required to unambiguously resolve novel full-length sequences. Overall, we identified 311 total full-length transcripts in a subset of 72 cynomolgus macaques from these Chinese breeding facilities; 130 of these sequences were novel and an additional 115 extended existing short database sequences to span the complete open reading frame. This significantly expands the number of Mafa-A, Mafa-B, and Mafa-I full-length alleles in the official cynomolgus macaque MHC class I database. The PacBio technique described here represents a general method for full-length allele discovery and genotyping that can be extended to other complex immune loci such as MHC class II, killer immunoglobulin-like receptors, and Fc gamma receptors.

INTRODUCTION

Cynomolgus macaques (Macaca fascicularis; Mafa) are native to much of continental and insular Southeast Asia, but there are no feral cynomolgus macaques living in China. However, Chinese breeding facilities are one of the largest exporters of cynomolgus macaques for use in biomedical research. According to the Centers for Disease Control and Prevention, China provided over 70% of the total cynomolgus macaques imported into the United States in fiscal year 2014. These animals are derived from other geographic populations of cynomolgus macaques, most likely from Thailand, Laos, Vietnam, Cambodia, and Malaysia, but also possibly including animals from Indonesia and the Philippines. Cynomolgus macaques from Chinese breeding facilities therefore are genetically heterogeneous, and this heterogeneity may be particularly biologically relevant in highly diverse genomic regions like the major histocompatibility complex (MHC).

The MHC is both highly complex and important – the gene products encoded by the MHC present peptides to T-cells, playing an essential role in immune response to pathogens and other non-self peptides (Bontrop 2006). In macaques, the MHC class I region is highly polymorphic and extensively duplicated (Daza-Vamenta et al. 2004; Otting et al. 2005), with different chromosomes carrying different numbers of functional Mafa-A and Mafa-B genes. Many investigators have examined the MHC class I region of cynomolgus macaques from various origins (Budde et al. 2010; Campbell et al. 2008; Kita et al. 2009; Lawrence et al. 2012; Ling et al. 2012; Otting et al. 2007, 2009, 2012; Pendley et al. 2008; Saito et al. 2012; Shiina et al. 2015; Uda et al. 2004, 2005; Zhang et al. 2012; Zhuo et al. 2011), but only a limited number of studies have looked at cynomolgus macaques from Chinese breeding facilities (Krebs et al. 2005; Westbrook et al. 2015).

Here we describe a comprehensive study of 100 samples collected from two different breeding facilities in China that include individuals reported to be of Vietnamese, Cambodian, and mixed Cambodian/Indonesian origins. Many of the samples were related to at least one other animal in the study, allowing us to characterize lineage-level haplotypes based on shared Illumina short-read transcript sequences. We then performed full-length MHC class I transcript analysis on a subset of the samples using the PacBio long-read single-molecule real-time (SMRT) sequencing system. This analysis improved on a previous MHC class I study using PacBio circular consensus SMRT sequencing (Westbrook et al. 2015) by using more advanced sequencing chemistry, and a data analysis pipeline focused on the highest quality reads that did not need to account for sequencing reads with insertion/deletion errors.

MATERIALS AND METHODS

Animals

A total of 100 whole blood samples were collected from two different breeding facilities in China. Thirty samples from reported Vietnamese-origin animals were obtained from Yulin Hongfeng Experimental Animal’s Domesticating and Breeding Center (Guangxi, China). A total of 70 samples were obtained from Hainan Jingang Laboratory Animal Co., Ltd. (Hainan, China) - 57 samples were obtained from reported Cambodian-origin animals, and 13 samples were from mixed Cambodian/Indonesian-origin animals. Blood sampling was performed under anesthesia in accordance with ethical guidelines of China. Samples were collected into TRIzol Reagent (ThermoFisher Scientific, Waltham, MA, USA) to stabilize the total RNA for shipment from China. Samples were requested from sire/dam/progeny trio sets to obtain samples that share MHC haplotypes that are identical by descent. Microsatellite genotyping was performed to assess accuracy of pedigree information (de Groot et al. 2014). Through this analysis, 29 total incorrect parental assignments were identified across both facilities. The remaining 40 sires and dams were correctly associated with the 31 progeny samples. The 71 total samples that were directly related to at least one other animal in the cohort were processed as sire/dam/progeny trio sets or parent/progeny duos; the remaining 29 samples were categorized as unrelated animals.

RNA isolation, cDNA synthesis, and PCR preparation

Total RNA was isolated from the blood samples and cDNA was produced from the isolated RNA using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific) following manufacturer’s protocols. For Illumina MiSeq genotyping, a 195 bp amplicon spanning the most polymorphic region of MHC class I molecules was generated using primers designed in conserved regions of exon 2, as shown in Supplemental Fig. 1. Amplification was performed using a 4-primer system to add unique barcodes to each sample, using consensus sequence adapters and barcodes designed by the Fluidigm Corporation (San Francisco, CA, USA), as detailed in Supplemental File 1.

For PacBio RS II full-length MHC class I transcript sequencing of 72 samples from the full cohort, an ~1,150 bp amplicon with primers located in the 5’UTR and 3’UTR of MHC class I sequences was generated as shown in Supplemental Fig. 2. A cocktail of two forward primers and three reverse primers was used to maximize homology with the majority of MHC class I sequences. A unique set of barcoded primers was used for each sample, with the barcodes integrated onto the ends of the sequence-specific oligos at synthesis. Barcodes are available from Pacific Biosciences (Menlo Park, CA, USA). Primer sequences are shown in Supplemental Fig. 2, and PCR conditions are detailed in Supplemental File 1.

All PCR products were purified using the AMPure XP PCR purification kit (Agencourt Bioscience Corporation, Beverly, MA, USA) and quantified using the Quant-iT dsDNA HS Assay kit and a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocols.

Sequencing

The MHC class I exon 2 genotyping amplicons were sequenced on an Illumina MiSeq instrument (San Diego, CA, USA), and the full-length MHC class I transcripts were sequenced on a PacBio RS II instrument (Menlo Park, CA, USA) following the respective manufacturer’s protocols. Additional sequencing details are provided in Supplemental File 1, and an overview of the PacBio sequencing process is shown in Supplemental Fig. 3.

Data analysis

Processing of the MHC class I exon 2 genotyping amplicon was performed as described previously for MHC class II amplicons (Karl et al. 2014) using a custom-built command line pipeline to merge reads, trim primers, and compare MiSeq reads against a database of known cynomolgus macaque transcripts. Mafa-A and Mafa-B lineage-level haplotypes were determined for each of the 100 samples essentially as previously described (Heimbruch et al. 2015; Karl et al. 2013), first examining the sire/dam/progeny trio sets, then the parent/offspring duos, to look for sets of co-inherited sequences. Haplotypes for the unrelated animals in this cohort were inferred based on similarity to those observed in the related sets of animals.

A custom semi-automated pipeline was also developed for analysis of the PacBio MHC class I full-length transcripts; an overview of this pipeline is shown in Supplemental Fig. 4. Full details of the pipeline are available in Supplemental File 1, and the scripts and tools used are publicly available (https://bitbucket.org/dholab/mhc-long-amplicon-allele-discovery-in-mafa-from-china). In brief, the PacBio reads of insert (the consensus sequence generated from all passes around the circular SMRTbell template) were processed to eliminate indel sequences, short reads, and chimeric sequences using the SMRT Analysis v2.3 Reads of Insert protocol. Processed reads perfectly matching known full-length cynomolgus macaque transcripts were removed prior to clustering by using bbmap (version 34) to map reads to known full-length allele sequences. Reads not identical to known allele sequences were clustered using USEARCH -cluster_fast (version v8.0.1517_i86osx32) and requiring 100% identity within clusters. Clusters supported by three or more identical full-length reads were then binned as either extensions of known partial cynomolgus macaque sequences or putative novel transcripts, and a genotyping table with read counts per sample was generated. All extension and putative novel sequences were manually validated using Geneious Pro v9.0 (Biomatters Limited, Auckland, New Zealand) to perform BLAST alignments and create BAM alignment files of the reads of insert against the validated transcripts. Validated sequences were submitted to GenBank and the Immuno Polymorphism Database for the Major Histocompatibility Complex genes of Non-Human Primates (IPD-MHC NHP) (http://www.ebi.ac.uk/ipd/mhc/nhp/index.html) (de Groot et al. 2012; Robinson et al. 2013) for official nomenclature.

Data availability

Primer sequences are listed in Supplemental Fig. 1 and 2. Lineage-level Mafa-A and Mafa-B haplotypes assigned to each animal from the MiSeq exon 2 data are available in Supplemental Table 1, and full MiSeq read counts per transcript per animal are available in Supplemental Fig. 5. Full resolution Mafa-A and Mafa-B haplotypes assigned to each of the 72 animals examined by PacBio sequencing are available in Supplemental Table 2, and full PacBio read counts per transcript per animal are available in Supplemental Fig. 6. Novel and extension full-length sequences as listed in Table 1 are available from GenBank (LN851916-LN852021, LN994391-LN994530, LN998188, KY047492).

Table 1.

Full-length transcripts observed by PacBio sequencing

Allele Name	Full Length Accession Number	# Animals	# Reads	ORF Length (bp)	Category	Longest Previous Length (bp)	Previous Accession Number(s)
Mafa-A1*001:01:02	LN851916	3	123	1098	extension	1062	FR682846
Mafa-A1*001:01:03	LN994391	1	31	1098	novel	-	-
Mafa-A1*001:02:01	LN998188	1	54	1098	extension	1039	AY958101
Mafa-A1*001:05	LN851917	2	51	1098	novel	-	-
Mafa-A1*002:02	LN994392	1	35	1098	extension	1003	AB447609
Mafa-A1*003:01	LN994393	1	48	1098	extension	1065	AM295829
Mafa-A1*003:03	LN994394	1	78	1098	extension	1062	FR682835, HQ113982, HQ131743
Mafa-A1*003:05	LN994395	1	73	1098	extension	544	HQ131758
Mafa-A1*003:06	LN851918	3	337	1098	novel	-	-
Mafa-A1*003:07	LN994396	1	53	1098	novel	-	-
Mafa-A1*004:02	LN994397	1	60	1098	extension	1003	AB448751, HQ113954, HQ131752
Mafa-A1*006:01:01	LN851919	7	528	1098	extension	1062	AY958098, FR682819
Mafa-A1*007:01	LN851920	2	627	1098	extension	1042	AY958089, HQ113975
Mafa-A1*007:06	LN994398	1	163	1098	extension	1065	FR691532, KP307886
Mafa-A1*008:01	KP307887	1	178	1098	extant	1098	AY958104, HQ113960, HQ131766, KP307887
Mafa-A1*010:04	EU203706	1	117	1098	extant	1098	EU203706
Mafa-A1*010:05	EU203707	1	115	1098	extant	1098	EU203707, FR686840
Mafa-A1*010:07	LN851921	1	50	1098	novel	-	-
Mafa-A1*015:01	LN851922	3	309	1098	extension	1039	AY958087
Mafa-A1*018:01	LN851923	2	337	1098	extension	1062	AY958090, FR682839
Mafa-A1*018:06	FM246489	2	279	1098	extant	1098	FM246489
Mafa-A1*018:08	LN994399	1	36	1098	novel	-	-
Mafa-A1*022:01	LN994400	1	17	1098	extension	1042	AY958096, LN994401
Mafa-A1*022:04	LN851924	5	634	1098	extension	822	HM236273, HQ113976, HQ131745
Mafa-A1*022:05	LN851925	1	141	1098	extension	1062	FR682826, HQ113977, HQ131770
Mafa-A1*022:06	KY047492	1	293	1098	extension	1062	FR682828, HQ131736
Mafa-A1*022:09:01	KP307910	1	58	1098	extant	1098	KP307910
Mafa-A1*022:09:02	LN851926	1	47	1098	novel	-	-
Mafa-A1*026:01	KP307888	3	306	1098	extant	1098	AY958119, HQ131773, KP307888
Mafa-A1*027:01	LN851927	6	705	1098	extension	1065	AY958103, KP307889
Mafa-A1*027:02	LN994402	1	41	1098	extension	1042	AY958113
Mafa-A1*027:03	LN994403	1	102	1098	extension	1003	AB447615
Mafa-A1*028:01	LN851928	1	57	1098	extension	1062	FR682824, HQ113969, HQ131769
Mafa-A1*028:03	LN994404	1	225	1098	novel	-	-
Mafa-A1*028:04	LN994405	1	65	1098	novel	-	-
Mafa-A1*028:05	LN994406	1	22	1098	novel	-	-
Mafa-A1*032:03:02	LN851929	2	195	1098	extension	1062	FR682840
Mafa-A1*033:02	LN851930	2	157	1098	novel	-	-
Mafa-A1*038:01	LN851931	8	894	1098	extension	1065	AM295825, AY958114, FR682847, HQ131737
Mafa-A1*040:01:01	LN851932	3	275	1089	extension	1062	AY958115, FR682842
Mafa-A1*040:03	LN994407	1	70	1089	extension	1042	AY958095
Mafa-A1*041:01	LN851933	3	350	1098	extension	1042	AY958116
Mafa-A1*042:01	LN994408	1	109	1098	extension	1039	AY958117
Mafa-A1*042:02	LN851934	1	85	1098	extension	1062	FR682858
Mafa-A1*043:01	LN994409	1	144	1098	extension	1062	AY958118, FR686841
Mafa-A1*043:10	KP307853	2	127	1098	extant	1098	KP307853, KP307884
Mafa-A1*043:11	LN994410	1	43	1098	novel	-	-
Mafa-A1*045:01	LN851935	1	107	1098	extension	1062	AY958088, AY958120, FR682843
Mafa-A1*045:03	LN994411	1	114	1098	novel	-	-
Mafa-A1*053:01	LN994412	1	57	1098	extension	1065	AB447562, AM295839, HQ113973, HQ131754
Mafa-A1*055:01	LN994413	1	108	1098	extension	1065	AM295840
Mafa-A1*055:02	LN994414	1	214	1098	novel	-	-
Mafa-A1*056:03	KP307890	5	428	1098	extant	1098	KP307890
Mafa-A1*057:02	LN994415	1	85	1098	extension	1065	KP307880
Mafa-A1*058:02	LN851936	2	194	1098	extension	1062	FR682836
Mafa-A1*060:04	LN851937	1	51	1098	extension	1003	AB447574
Mafa-A1*063:03:01	LN851938	2	162	1098	extension	1062	AY958099, FR682832
Mafa-A1*064:03	LN994416	1	287	1098	extension	1003	AB447608
Mafa-A1*065:02	LN851939	1	36	1098	extension	1003	AB447611
Mafa-A1*065:03	LN851940	3	144	1098	extension	1062	AB447614, FR682853
Mafa-A1*065:04:01	LN851941	1	71	1098	extension	1062	AB448748, FR682825, FR682850
Mafa-A1*065:05	LN994417	1	59	1098	novel	-	-
Mafa-A1*066:06	LN851942	1	102	1098	extension	721	HQ113962, HQ131765
Mafa-A1*067:01	KP307908	4	584	1098	extant	1098	AM295855, FR682827, KP307908
Mafa-A1*070:01	EU203708	2	74	1098	extant	1098	AM295858, EU203708, FR682831
Mafa-A1*074:01:01	LN851943	2	220	1098	extension	1003	AB447596
Mafa-A1*077:01:02	LN994418	1	81	1098	extension	1003	AB447613
Mafa-A1*077:02:01	LN994419	1	101	1098	novel	-	-
Mafa-A1*077:02:02	LN994420	1	275	1098	novel	-	-
Mafa-A1*078:01:01	AB154767	1	111	1098	extant	1098	AB154767
Mafa-A1*085:02	LN851944	2	198	1098	extension	1062	FR682820
Mafa-A1*086:01:02	LN851945	2	12	981	novel	-	-
Mafa-A1*086:03	LN994421	1	5	981	extension	822	GU130454
Mafa-A1*086:05	LN994422	1	5	981	novel	-	-
Mafa-A1*089:06	LN851946	1	68	1098	extension	1003	AB447610
Mafa-A1*090:02:02	LN994423	1	10	1098	novel	-	-
Mafa-A1*090:03	LN851947	4	19	1098	novel	-	-
Mafa-A1*092:01	LN994424	1	41	1098	extension	1062	AB447594, FR682823, FR682833, HQ113961, HQ131768
Mafa-A1*092:03	LN994425	1	123	1098	extension	1065	FM246488
Mafa-A1*092:04	KP307869	1	103	1098	extant	1098	KP307869
Mafa-A1*095:02	LN994426	1	99	1098	novel	-	-
Mafa-A1*096:02	LN994427	2	129	1098	novel	-	-
Mafa-A1*097:02	LN994428	1	119	1098	novel	-	-
Mafa-A1*101:02:02	LN851948	1	67	1098	novel	-	-
Mafa-A1*102:03:01	LN851949	3	256	1098	novel	-	-
Mafa-A1*102:03:02	LN994429	1	146	1098	novel	-	-
Mafa-A1*124:01	LN994430	1	63	1098	extension	641	AB583237
Mafa-A1*130:01	LN851950	1	66	1098	extension	1080	KP307844
Mafa-A2*01:01	LN994431	1	62	1098	extension	1062	FR682848, HQ113963, HQ131750
Mafa-A2*05:06:02	LN994432	4	6	1098	extension	822	EF550522
Mafa-A2*05:06:04	LN994438	2	14	1098	novel	-	-
Mafa-A2*05:22	LN851952	2	14	1101	extension	822	EF589357
Mafa-A2*05:25:01	LN994433	5	16	1098	extension	822	EF589360, HP823075
Mafa-A2*05:31	LN851953	2	18	1101	extension	822	EF550520
Mafa-A2*05:40	LN994434	2	5	1098	extension	822	GQ131777
Mafa-A2*05:42	LN994435	1	20	1083	extension	822	GQ131775
Mafa-A2*05:45	LN994436	1	4	1098	extension	822	GQ131771
Mafa-A2*05:46	LN851954	2	9	1101	extension	1065	GQ131770, HQ230581
Mafa-A2*05:50	GU063713	1	1	1098	extant	1098	GU063713, LC053847
Mafa-A2*05:51	LN851955	1	3	1101	extension	822	GU130445
Mafa-A2*05:55	LN851956	5	48	1101	extension	1080	KP307855
Mafa-A2*05:57:01	LN851951	6	34	1098	extension	822	HQ992789
Mafa-A2*05:58	LN994437	1	5	1098	novel	-	-
Mafa-A2*05:59	LN994439	2	6	1098	novel	-	-
Mafa-A2*24:07	LN851957	1	49	1098	novel	-	-
Mafa-A2*24:08	LN994440	1	211	1098	novel	-	-
Mafa-A3*13:02	LN851958	2	27	1098	extension	1042	AY958111
Mafa-A3*13:07	LN851959	4	55	1098	extension	1062	FR682834, GQ131768, HM161349, HQ113968, HQ131739
Mafa-A3*13:14N	LN851960	2	5	1011	extension	822	GU130464
Mafa-A3*13:15	LN994441	1	6	1098	extension	641	AB583238
Mafa-A3*13:17:02	LN851961	1	5	1098	novel	-	-
Mafa-A3*13:20	LN851962	1	15	1098	novel	-	-
Mafa-A3*13:21	LN994442	1	6	1098	novel	-	-
Mafa-A3*13:22	LN994443	1	14	1098	novel	-	-
Mafa-A4*01:02	LN851963	2	9	1098	extension	1042	AY958121, JF729454
Mafa-A4*01:08	LN994444	6	11	1011	extension	822	GQ131766
Mafa-A4*01:12	LN994445	1	6	1011	novel	-	-
Mafa-A4*14:01	LN851964	9	31	1011	extension	1065	AM295880
Mafa-A4*14:03	LN851965	21	110	1101	extension	1065	FR682829, GQ131781, HQ113965, HQ131747, LC043320
Mafa-A4*14:04	LN851967	1	3	1101	extension	822	GQ131767
Mafa-A4*14:13	KP307856	1	22	1092	extant	1092	KP307856
Mafa-A4*14:15	LN851968	1	6	1107	novel	-	-
Mafa-A4*14:16	LN851966	1	3	1101	novel	-	-
Mafa-A4*14:18	LN994446	1	4	1107	novel	-	-
Mafa-A4*14:19	LN994447	1	7	1101	novel	-	-
Mafa-A5*30:06	KP307871	1	1	1089	extant	1089	KP307871
Mafa-B*001:01:01	KP307893	7	239	1089	extant	1089	AY958126, HQ131715, KP307893
Mafa-B*001:01:02	LN994449	1	54	1089	novel	-	-
Mafa-B*001:02	LN994448	1	19	1089	novel	-	-
Mafa-B*002:04	LN994450	3	472	1089	extension	822	HM161352, HQ114001, HQ131729, HQ131749
Mafa-B*006:01:02	LN994451	1	12	1089	novel	-	-
Mafa-B*007:01:01	GU063753	17	606	1089	extant	1089	AY958137, GU063753, HM161384, HM236279, HQ131730, LC043321
Mafa-B*007:01:05	LN851969	6	155	1089	extension	822	HM161391, HQ114005, HQ131716
Mafa-B*007:05	LN851970	4	98	1089	extension	544	HQ131707
Mafa-B*007:06:02	LN994453	1	48	1089	novel	-	-
Mafa-B*007:07	LN851971	3	53	1089	novel	-	-
Mafa-B*007:08	LN851972	7	235	1089	novel	-	-
Mafa-B*007:10	LN994452	1	27	1089	novel	-	-
Mafa-B*008:01	LN994454	1	56	1089	extension	544	HQ131700
Mafa-B*010:01	AB195452	2	112	1089	extant	1089	AB195452
Mafa-B*011:01	AY958143	1	42	1089	extant	1089	AY958143, EU203717, GU063737
Mafa-B*013:03	LN994455	2	45	1089	extension	822	FJ178813
Mafa-B*013:06	LN851973	1	32	1089	extension	822	GQ131788, HQ113998, HQ131703
Mafa-B*013:09	KP307894	3	269	1089	extant	1089	FJ178814, HQ131706, KP307894
Mafa-B*013:10	LN851974	2	42	1089	extension	1056	HM161366, HQ131722, KP307895
Mafa-B*013:11	LN994456	1	45	1089	extension	1056	FM212794
Mafa-B*013:13	LN851975	1	27	1089	novel	-	-
Mafa-B*013:14	LN851976	2	104	1089	novel	-	-
Mafa-B*013:15	LN994457	1	44	1089	novel	-	-
Mafa-B*013:16	LN994458	1	118	1089	novel	-	-
Mafa-B*013:17	LN994459	1	18	1089	novel	-	-
Mafa-B*013:18	LN994460	1	4	1089	novel	-	-
Mafa-B*014:01	LN851977	2	14	1005	extension	1056	FM212831
Mafa-B*014:02	LN851978	1	8	1005	novel	-	-
Mafa-B*015:01	LN851979	2	49	1089	extension	1042	AY958132, HM235701
Mafa-B*015:05	LN994461	1	42	1089	novel	-	-
Mafa-B*015:06	LN994462	1	106	1089	novel	-	-
Mafa-B*016:01	EU203701	1	118	1092	extant	1092	EU203701
Mafa-B*016:02	LN994463	1	61	1092	novel	-	-
Mafa-B*017:02	KP307896	2	43	1080	extant	1080	HQ131701, KP307896
Mafa-B*018:01:01	KP307897	3	178	1089	extant	1089	AY958138, KP307897
Mafa-B*018:03	LN994464	1	13	1089	novel	-	-
Mafa-B*019:03	DQ979879	1	7	1086	extant	1086	DQ979879, GU063743
Mafa-B*019:04	LN994465	1	33	1086	novel	-	-
Mafa-B*021:02	KP307859	8	127	1077	extant	1077	KP307859, KP307845
Mafa-B*021:04	LN994466	1	10	1077	novel	-	-
Mafa-B*021:05	LN994467	2	37	1077	novel	-	-
Mafa-B*025:02	LN851980	1	110	1086	novel	-	-
Mafa-B*028:02	KP307898	6	18	1089	extant	1089	AY958130, KP307898
Mafa-B*028:03	LN994468	1	6	1089	extension	1042	AY958128, HQ131724, LC043325
Mafa-B*028:04	EU046324	2	20	1089	extant	1089	EU046324, HM235716, HQ131708
Mafa-B*030:01:01	LN851983	7	277	1089	extension	1042	AY958133, HQ131709
Mafa-B*030:01:05	LN994472	2	22	1089	novel	-	-
Mafa-B*030:02	LN994469	1	16	1089	extension	1042	AY958134, HQ131767
Mafa-B*030:03:01	KP307900	2	10	1089	extant	1089	AY958135, HQ131687, KP307900
Mafa-B*030:03:02	LN994470	5	18	1089	extension	822	FJ719167, HQ131726
Mafa-B*030:04	LN994471	1	48	1089	extension	822	FJ719169
Mafa-B*030:15	KP307857	2	34	1089	extant	1089	KP307857
Mafa-B*030:16	LN851984	2	22	1089	novel	-	-
Mafa-B*030:17	KP307899	5	28	1089	extant	1089	FJ178822, KP307899
Mafa-B*030:18	LN994473	2	5	1089	novel	-	-
Mafa-B*030:19	LN994474	1	4	1089	novel	-	-
Mafa-B*031:03	LN994475	2	21	1095	novel	-	-
Mafa-B*034:01	LN851985	1	62	1089	extension	1042	AY958127
Mafa-B*034:03	LN851986	2	131	1089	extension	819	HQ113997, HQ131704
Mafa-B*034:04	LN994476	1	34	1089	extension	1056	KP307879
Mafa-B*036:01:02	EU606046	2	39	1080	extant	1080	EU606046, FM212796, HQ131685
Mafa-B*037:01	AY958149	2	22	1089	extant	1089	AY958149, HQ114004, HQ131697
Mafa-B*038:01	KP307860	2	31	1089	extant	1089	KP307860
Mafa-B*039:01	KP307902	11	674	1089	extant	1089	HM161344, HQ113949, HQ113992, HQ114010, HQ131713, HQ131762, KP307902
Mafa-B*039:02	LN994477	2	87	1089	novel	-	-
Mafa-B*041:02	KP307858	4	28	1089	extant	1089	KP307858, KP307874, KP307882, LC053850
Mafa-B*041:03	LN851987	1	9	1089	extension	1071	KP307846
Mafa-B*043:01	LN994478	2	33	1089	extension	646	AB569230
Mafa-B*043:02	LN994479	1	15	1089	novel	-	-
Mafa-B*044:01:01	KP307903	10	113	1080	extant	1080	FM212817, HM235712, KP307903
Mafa-B*044:01:03	LN994480	1	43	1080	novel	-	-
Mafa-B*044:04	HM581964	1	47	1080	extant	1080	HM581964
Mafa-B*045:07	LN994481	2	21	1089	novel	-	-
Mafa-B*047:03	LN994482	1	53	1089	novel	-	-
Mafa-B*048:06	LN851988	1	54	1089	novel	-	-
Mafa-B*050:02:02	LN994483	1	15	1080	novel	-	-
Mafa-B*050:05	LN851989	5	55	1080	extension	546	HM161343, HQ131714
Mafa-B*050:08	LN851990	2	10	1080	extension	1003	LC043337
Mafa-B*056:01	KP307904	10	717	1089	extant	1089	AY958131, HQ131699, KP307904
Mafa-B*056:04	KP307848	1	29	1089	extant	1089	KP307848
Mafa-B*056:05:01	LN994484	1	52	1089	extension	1056	HM235703, HQ131733, KP307905
Mafa-B*056:05:02	LN994485	1	54	1089	novel	-	-
Mafa-B*060:01	EU203692	4	6	1080	extant	1080	EU203692
Mafa-B*060:04	HM581968	5	12	1080	extant	1080	AB569226, HM581968, HQ131693
Mafa-B*060:12	LN851991	1	3	1080	novel	-	-
Mafa-B*060:13	LN851992	12	57	1080	novel	-	-
Mafa-B*060:14	LN994487	1	5	1080	novel	-	-
Mafa-B*060:16	LN994486	1	8	1080	novel	-	-
Mafa-B*061:03:01	KP307851	2	48	1080	extant	1080	KP307851
Mafa-B*061:03:02	LN851993	1	11	1080	novel	-	-
Mafa-B*061:04:01	KP307861	6	270	1080	extant	1080	KP307861, KP307843
Mafa-B*061:04:02	LN851994	2	77	1080	novel	-	-
Mafa-B*061:05	LN994488	2	5	1089	novel	-	-
Mafa-B*064:05	LN994489	1	4	1077	novel	-	-
Mafa-B*065:02	LN851995	3	28	1089	extension	822	HM161351, HQ113986, HQ131727
Mafa-B*065:04	LN851996	5	70	1089	novel	-	-
Mafa-B*065:05	LN994490	1	40	1089	novel	-	-
Mafa-B*066:01	KP307849	1	16	1089	extant	1089	KP307849
Mafa-B*067:01	LN994491	1	47	1089	novel	-	-
Mafa-B*067:02	LN994492	1	31	1089	novel	-	-
Mafa-B*068:02	FM212839	4	79	1089	extant	1089	FM212839
Mafa-B*068:04	LN851997	7	110	1089	extension	641, 1056	AB569236, KP307906
Mafa-B*068:06:01	KP307865	6	120	1089	extant	1089	KP307865
Mafa-B*068:06:02	LN994493	1	23	1089	novel	-	-
Mafa-B*068:07:01	KP307850	1	20	1089	extant	1089	KP307850
Mafa-B*068:07:02	LN994494	2	57	1089	novel	-	-
Mafa-B*068:10	LN851998	1	5	1089	novel	-	-
Mafa-B*069:03	LN852001	3	144	1089	extension	544	HQ131732
Mafa-B*069:04	LN851999	2	113	1089	extension	822	HM161434, HQ114002, HQ131710
Mafa-B*069:05	LN852000	2	152	1089	novel	-	-
Mafa-B*069:06	LN994495	1	90	1089	novel	-	-
Mafa-B*069:07	LN994496	1	28	1089	extension	822	HM161424
Mafa-B*070:04	LN852002	2	10	1080	extension	1070	KP307842
Mafa-B*072:02	HM581961	1	3	1089	extant	1089	HM581961
Mafa-B*072:04	LN852003	2	5	1089	novel	-	-
Mafa-B*073:02	KP307876	2	82	1089	extant	1089	HM161413, KP307876
Mafa-B*073:03	LN994497	1	18	1089	novel	-	-
Mafa-B*074:02	LN852004	7	132	1080	extension	646	AB569228
Mafa-B*075:01	EU203718	1	73	1089	extant	1080	AY958150, EU203718, GU063741
Mafa-B*077:01	FM212843	1	88	1089	extant	1089	FM212843
Mafa-B*077:02	LN852005	1	59	1089	novel	-	-
Mafa-B*079:01:01	AM943362	1	1	1080	extant	1080	AM943362
Mafa-B*079:02:01	LN994499	5	9	1080	extension	822	HM236276
Mafa-B*081:01	LN852006	2	31	1086	extension	822	AB569225, HM161374, HQ131721
Mafa-B*082:03	LN994500	6	20	1116	novel	-	-
Mafa-B*083:02	KP307907	1	151	1089	extant	1089	HM236289, KP307907
Mafa-B*083:04	LN994501	1	16	1089	novel	-	-
Mafa-B*085:01	KP307852	7	214	1089	extant	1089	AY958124, HM161375, HQ113995, HQ131720, KP307852, LC043363
Mafa-B*088:04	LN852007	8	13	1089	novel	-	-
Mafa-B*088:05	LN994502	6	12	1089	novel	-	-
Mafa-B*089:01:02	EU392125	6	18	1116	extant	1116	EU392125, FJ178818, HM161360, HQ113989, HQ131731, JF729417, LC043349
Mafa-B*091:03	LN994503	1	70	1089	extension	822	HM161370
Mafa-B*093:02	LN852008	5	242	1089	extension	822	HM161359, HQ113988, HQ114006, HQ131734
Mafa-B*095:03	LN994504	1	12	1089	novel	-	-
Mafa-B*095:04	LN994505	1	173	1089	novel	-	-
Mafa-B*098:03:02	LN994508	3	5	1089	novel	-	-
Mafa-B*098:04	LN994506	3	6	1089	extension	981	HM581969
Mafa-B*098:12	LN994507	6	15	1089	novel	-	-
Mafa-B*101:02	LN994509	2	164	1089	extension	819	HQ113999, HQ131702
Mafa-B*103:01:02	LN994510	1	6	1080	novel	-	-
Mafa-B*104:01:02	FM212841	6	698	1089	extant	1089	FM212841, HQ131684, HQ131774
Mafa-B*105:01	LN852009	2	80	1089	extension	819	HQ114013, HQ131692
Mafa-B*110:01:01	LN852010	4	162	1089	extension	822	HM161339, HM161378, HQ131725
Mafa-B*110:01:02	LN994511	1	13	1089	novel	-	-
Mafa-B*117:03	LN852011	2	26	1080	novel	-	-
Mafa-B*117:04	LN994512	1	18	1080	novel	-	-
Mafa-B*124:01:01	EU203714	1	27	1080	extant	1080	EU203714
Mafa-B*124:01:03	KP307862	6	105	1080	extant	1080	KP307862, KP307847
Mafa-B*124:03	LN852012	2	9	1080	novel	-	-
Mafa-B*124:04	LN994513	1	6	1080	novel	-	-
Mafa-B*137:03	EU203723	6	138	1089	extant	1089	EU203723, EU392117, HM235700, HQ131688, HQ131740, JF729416, LC043364
Mafa-B*137:05	LN852013	1	12	1089	novel	-	-
Mafa-B*137:06	LN994515	2	32	1089	novel	-	-
Mafa-B*137:07	LN994514	1	4	1089	novel	-	-
Mafa-B*137:08	LN994516	1	11	1089	novel	-	-
Mafa-B*139:02	FM212840	2	19	1086	extant	1086	FM212840
Mafa-B*140:01:02	LN994517	1	27	1089	novel	-	-
Mafa-B*144:01	LN994518	2	38	1080	extension	1042	AY958122, HQ113983, HQ131682
Mafa-B*145:01	LN852014	3	198	1089	extension	1042	AY958123, HM235702, HQ113984, HQ131728
Mafa-B*146:01:01	LN852015	1	24	1089	extension	1042	AY958125
Mafa-B*148:01:01	LN994519	1	22	1089	novel	-	-
Mafa-B*149:01	EU606047	2	4	1089	extant	1089	EU606047
Mafa-B*149:02	LN852016	2	10	1089	extension	822	HG977501, HQ110973
Mafa-B*150:02	LN994520	1	96	1089	extension	1056	FM212814
Mafa-B*155:01	EU203684	1	45	1080	extant	1080	EU203684, HM235713
Mafa-B*156:01	EU203694	2	57	1089	extant	1089	EU203694, FJ178816
Mafa-B*161:05	LN852017	2	57	1089	novel	-	-
Mafa-B*164:02	GQ153333	1	49	1089	extant	1089	GQ153333, LC043371
Mafa-B*181:03	LN994521	1	41	1089	novel	-	-
Mafa-B*184:01:01	LN994522	2	162	1089	extension	822	JN032108
Mafa-B*184:01:02	LN994523	2	100	1089	novel	-	-
Mafa-B*203:01	LN994498	1	81	1089	novel	-	-
Mafa-I*01:01:02	LN994530	1	9	1089	novel	-	-
Mafa-I*01:09	AB195465	1	2	1089	extant	1089	AB195465
Mafa-I*01:11:02	LN852020	14	46	1089	novel	-	-
Mafa-I*01:14:02	LN852021	4	5	1089	novel	-	-
Mafa-I*01:18:01	LN994524	8	34	1089	extension	1056	FM246497
Mafa-I*01:22	HM581970	1	2	1089	extant	1089	AF161864, HM581970
Mafa-I*01:29	LN852018	2	6	1089	novel	-	-
Mafa-I*01:30	LN852019	4	4	1089	novel	-	-
Mafa-I*01:31	LN994525	4	6	1089	novel	-	-
Mafa-I*01:32	LN994526	3	6	1089	novel	-	-
Mafa-I*01:33	LN994527	4	8	1089	novel	-	-
Mafa-I*01:34	LN994528	1	6	1089	novel	-	-
Mafa-I*01:35	LN994529	1	6	1089	novel	-	-

Summary of 311 full-length MHC class I transcripts identified in 72 cynomolgus macaques from Chinese breeding facilities. Allele names and accession numbers in bold font are novel to this study or add sequence to an existing named allele to extend it to full-length

RESULTS AND DISCUSSION

Lineage-level haplotype analysis

In human and macaque MHC class I nomenclature, closely related alleles originating from the same gene are assigned to the same lineage group, signified by the 2–3 digits between the asterisk and the first colon of MHC class I sequence names. Since the Illumina MiSeq exon 2 genotyping amplicon is focused on a small segment of the MHC class I molecule, it is generally only sufficient for determining the lineage groups of any detected MHC class I sequences (considered ‘three-digit’ resolution in human HLA genotyping and referred to as ‘lineage-level’ resolution here). However, it provides a rapid, high-throughput methodology for haplotype analysis, especially within related animals. Using the parent/progeny trio and duo sets within these two cohorts, we described haplotypes of Mafa-A and Mafa-B transcripts inherited together from parents to offspring. Two representative trio sets with parents and their progeny are illustrated in Fig. 1a. Any transcripts observed as shared between parent and offspring were assigned to the inherited haplotype; any remaining transcripts observed within a parent, or in an offspring with just one representative parent, were assigned to the alternate haplotype. For animals without a direct relative in the cohort, haplotypes were inferred based on identity to the defined inherited haplotypes. We focused on the ‘major’ transcripts with high levels of steady state RNA for haplotype definitions. Abbreviated haplotype names were assigned based on presence of a major ‘diagnostic’ sequence that was typically the most abundant transcript on the haplotype, i.e., B013 haplotypes express the diagnostic major transcript Mafa-B*013. Different combinations of major allele lineages inherited with each diagnostic transcript were denoted with lowercase letters following the haplotype name, e.g., Mafa-B*013 inherited with Mafa-B*007 was designated B013a and Mafa-B*013 inherited with Mafa-B*137 was designated B013d. The full list of Mafa-A and Mafa-B haplotypes defined in these cohorts of cynomolgus macaques from Chinese breeding facilities is given in Supplemental Table 1.

Fig. 1.

Representative trio set results by a) MiSeq genotyping and b) PacBio full-length sequencing methods. The major transcripts for two representative trio sets are shown here, with progeny located between the sire and dam, showing direct transcript inheritance from parents to offspring. Inherited haplotypes are outlined in solid boxes. Groups of Mafa-A and Mafa-B transcripts co-inherited together as a haplotype are colored correspondingly. Transcripts shown in bold font are diagnostic major sequences used to name the haplotypes. Transcripts shown in grey font were only observed by MiSeq. Values indicate the number of reads for each sequence per sample

A total of 52 distinct Mafa-A haplotypes and 74 unique Mafa-B haplotypes were identified in the 100 animals (200 chromosomes) evaluated in this cohort (Supplemental Table 1). A summary of the lineage-level haplotypes observed in each animal is shown in Fig. 2, with haplotypes directly inherited from parent to offspring denoted with solid outlined boxes. Full MiSeq haplotyping results, including reads supporting each transcript call per sample, are also given in Supplemental Fig. 5. While overall the large number of related animals in the cohort skews haplotype frequencies, it should be noted that four Mafa-A haplotypes (A006, A022a, A027, and A065) and three Mafa-B haplotypes (B013d, B028d, and B039e) are each observed in ten or more individuals. There are 1–2 major alleles expressed on each Mafa-A haplotype, accompanied by 0–5 minor alleles. Each Mafa-B haplotype is associated with 1–7 expressed major transcripts and between 0–15 minor transcripts.

Fig. 2.

Summary of MHC class I Mafa-A and Mafa-B haplotypes observed in each macaque. Samples are organized by geographic origin into family groups, and directly inherited haplotypes are outlined with solid boxes. The full list of transcripts associated with each haplotype are available in Supplemental Table 1

Though many studies have examined the lists of MHC class I transcripts expressed in cynomolgus macaques of multiple geographic origins, few have studied the haplotype structures in those animals (Budde et al. 2010; Campbell et al. 2008; Otting et al. 2009, 2012; Saito et al. 2012; Shiina et al. 2015). The best-characterized population of cynomolgus macaques is from the island of Mauritius, where a limited number of founding animals were deposited on the island in the 1500s. Since the entire current population arose from a small number of ancestors, the diversity of their MHC region (and their entire genome) is limited to five lineage-level Mafa-A haplotypes (A031, A032a, A033, A060, A063) and seven lineage-level Mafa-B haplotypes (B019a, B045a, B075a, B104a, B147, B151a, B164a) (Budde et al. 2010; Wiseman et al. 2013). The diversity observed in these cohorts of 100 individuals from two Chinese breeding facilities represents a ten-fold increase in diversity at each locus compared to Mauritian cynomolgus macaques. While both populations of cynomolgus macaques are popular for biomedical research, this difference in overall diversity suggests that investigators should carefully consider their objectives when choosing a source for their animals. For protocols where MHC may be a confounding factor, like SIV and transplantation research, the restricted diversity of the Mauritian cynomolgus macaque population is particularly attractive. When testing vaccines or other drug therapies for potential negative side effects however, an extremely diverse source like cynomolgus macaques from Chinese breeding facilities may be advantageous. The overall diversity observed in this cohort also suggests that researchers should have their animals haplotyped if the MHC is a possible modifying factor, since this study revealed a large number of haplotypes despite the fact that multiple related animals were evaluated. Lineage-level MiSeq haplotype analysis should be sufficient for most researchers looking to balance haplotypes across experimental and control cohorts or remove MHC haplotypes with known confounding alleles from their studies.

This study likely represents a snapshot of the total MHC diversity available within cynomolgus macaques from Chinese facilities, largely dependent on the country of origin of their breeding animals. Of the 52 total Mafa-A haplotypes observed here, nine (17%) were observed exclusively in animals of reported Vietnamese heritage, 17 (33%) were of reported Cambodian-origin only, and 4 (8%) were observed in the small set of reported hybrid Cambodian/Indonesian-origin animals (Supplemental Table 1). Only 22 (42%) Mafa-A haplotypes were observed in samples from multiple origins. The results for the 74 Mafa-B haplotypes are even more striking with only 21 (28%) observed in samples from multiple origins. Among the remaining Mafa-B haplotypes, 14 (19%) were exclusive to Vietnamese-origin animals, 26 (35%) exclusive to Cambodian-origin, and 13 (18%) were observed only in the mixed Cambodian/Indonesian-origin individuals. No previous MHC haplotype studies have been performed in either Vietnamese- or Cambodian-origin cynomolgus macaques, but limited studies have characterized haplotypes in Filipino, Indonesian, and Malaysian cynomolgus macaques (Campbell et al. 2008; Otting et al. 2009, 2012; Saito et al. 2012; Shiina et al. 2015). The cohorts studied here appear more MHC diverse than those sampled from the previously described insular and extremely southern continental Asian populations (it is unclear from precisely where the Malaysian cohort was initially derived). It should be noted, however, that these cohorts were requested to be drawn from different breeding groups within each facility to maximize potential for novel allele discovery, so it is difficult to draw any conclusions about the overall degree of inbreeding or influence of prolific sires on the MHC genetics of Chinese breeding facilities as a whole.

Full-length allele discovery

In this study we employed a novel method for next-generation sequencing of full-length MHC class I alleles, using the PacBio RS II SMRT long read sequencing instrument. In our previous study with P4-C2 PacBio sequencing (Westbrook et al. 2015), data analysis relied on creation of contigs at 99% identity (largely to account for insertion/deletion artifacts) to define transcripts. Since this initial study, advances in PacBio sequencing with P6-C4 reagents and analysis software have eliminated the need to include less than 100% identical reads for identification of novel sequences. From the full 100 animal cohort, we selected 72 total samples across both breeding centers for PacBio sequencing based on lineage-level haplotype diversity. Table 1 presents a complete list of all 311 full-length MHC class I transcripts that we detected in these 72 individuals; these include 126 Mafa-A, 172 Mafa-B, and 13 Mafa-I transcripts. The complete PacBio sequencing results for each animal including numbers of identical sequencing reads supporting each transcript call are shown in Supplemental Fig. 6. Overall, nearly 80% of the transcripts identified in this study added new information to the existing Mafa MHC class I allele databases – 130 (42%) of the sequences were completely novel to the database, and 115 (37%) extended sub-full length but previously named database sequences to cover full-length open reading frames.

This study improved the extant cynomolgus macaque allele databases in both total number of sequences and, more importantly, in the percentage of known transcripts that have been unambiguously described over the full coding region. The November 2015 update of IPD-MHC NHP contained a total of 899 Mafa-A, Mafa-B, and Mafa-I sequences. Of these, only 386 (43%) were full-length from start codon to stop codon. This study adds an additional 130 novel transcripts to the database, bringing the total to 1,029 named MHC class I sequences in the cynomolgus macaque database. With the additional 115 extant sequences extended to full length, the database of Mafa-A, Mafa-B, and Mafa-I transcripts following this study now contains 631 full-length sequences (61% of the 1,029 total transcripts).

Various geographical origins have been reported for the 181 total extant and extended sequences observed in this study (Table 2). This is an under-representation of observed origins for these sequences since not all studies reported geographical origins for their cynomolgus macaques (including previous studies examining cynomolgus macaques from Chinese breeding facilities). This is the first study of MHC alleles in Cambodian- and Cambodian/Indonesian-hybrid origin animals, so all geographical origin associations for the transcripts from these animals is new to this study. The majority of the transcripts observed in the Vietnamese-origin animals had been previously reported in other studies of Vietnamese cynomolgus macaques; however, twelve sequences from Vietnamese-origin animals in our study had only been observed previously in Indonesian, Mauritian, Malaysian, or Indonesian/Malaysian hybrid animals (an additional eight sequences in our Vietnamese-origin animals had no previously reported origins). Overall, this indicates that MHC class I allele sharing between cynomolgus macaques of various geographical origins may be more common than previously appreciated.

Table 2.

Cynomolgus macaque transcript geographical origins and identity to rhesus and pig-tailed macaque sequences

Mafa Allele Name	Origin(s)	Previous Mafa Geographical Origin(s)	Origin Reference(s)	Identical To
Mafa-A1*001:01:02	H	?	1
Mafa-A1*001:02:01	V	V	2,3	Mamu-A1*001:03 (LN899620 – 1098bp)
Mafa-A1*002:02	C	V	3
Mafa-A1*003:01	H	U	4
Mafa-A1*003:03	C	V	1,5	Mamu-A1*003:01:01 (U41379 – 1014bp)
Mafa-A1*003:05	C	V	5
Mafa-A1*003:07	V	-	-	Mamu-A1*003:05 (LN899624 – 1098bp)
Mafa-A1*004:02	V	V	3,5	Mamu-A1*004:01:01 (FN396407 – 1098bp)
Mafa-A1*006:01:01	C, V	A, V	1,2
Mafa-A1*007:01	C, V	A, V	2,5
Mafa-A1*007:06	C	A	1,6	Mamu-A1*007:01 (U41832 – 1098bp)
Mafa-A1*008:01	C	A, V	2,5,6
Mafa-A1*010:04	H	I	7
Mafa-A1*010:05	C	I, V	1,3,7
Mafa-A1*015:01	C, V	V	2,3	Mamu-A1*015:01 (AB551785 – 649bp)
Mafa-A1*018:01	C, V	V	1,2
Mafa-A1*018:06	H	I, U	8,9
Mafa-A1*022:01	C	V	2
Mafa-A1*022:04	V, H	V	5,10
Mafa-A1*022:05	V	V	1,5
Mafa-A1*022:06	C	V	1,5
Mafa-A1*022:09:01	C	A	6
Mafa-A1*026:01	C	A, V	2,5,6
Mafa-A1*027:01	C, V	A, V	2,6
Mafa-A1*027:02	V	A	2
Mafa-A1*027:03	V	V	3
Mafa-A1*028:01	C	V	1,5
Mafa-A1*028:03	C	-	-	Mamu-A1*028:03 (LN899634 – 1098bp)
Mafa-A1*028:05	C	-	-	Mamu-A1*028:07 (HM594291 – 1098bp)
Mafa-A1*032:03:02	C	?	1
Mafa-A1*033:02	V	-	-	Mamu-A1*033:02 (JQ866400 – 1098bp)
Mafa-A1*038:01	C, H, V	A, U, V	1,2,3,4,5
Mafa-A1*040:01:01	C	A	1,2	Mamu-A1*040:01 (AM295910 – 1065bp)
Mafa-A1*040:03	V	V	2
Mafa-A1*041:01	C	A, V	2,3	Mamu-A1*041:02:01 (FN985475 – 1098bp)
Mafa-A1*042:01	C	A, V	2,3
Mafa-A1*042:02	V	?	1
Mafa-A1*043:01	V	A, V	1,2,3	Mamu-A1*043:06 (GU592078 – 1086bp)
Mafa-A1*043:10	V	A	6
Mafa-A1*045:01	V	A, V	1,2	Mamu-A1*045:01 (EU262741 – 1065bp)
Mafa-A1*053:01	C	U, V	3,4,5	Mamu-A1*053:01 (AM295918 – 1065bp)
Mafa-A1*055:01	H	U	4
Mafa-A1*056:03	C, V	A	6	Mamu-A1*056:02:01 (AM295922 – 1098bp)
Mafa-A1*057:02	C	A	6	Mamu-A1*057:01 (EU682514 – 1098bp)
Mafa-A1*058:02	C, H	?	1
Mafa-A1*060:04	V	I	3
Mafa-A1*063:03:01	C	V	1,2	Mamu-A1*063:01 (LN899635 – 1098bp)
Mafa-A1*064:03	V	V	3
Mafa-A1*065:02	V	V	3
Mafa-A1*065:03	C, V	V	1,3
Mafa-A1*065:04:01	C	V	1,3	Mamu-A1*065:01 (EU418506,KF855146 – 1098bp)
Mafa-A1*066:06	V	V	5	Mamu-A1*066:01 (AB444888 – 657bp)
Mafa-A1*067:01	C	A, U	1,4,6
Mafa-A1*070:01	V	I, U, V	1,3,4,7
Mafa-A1*074:01:01	V	V	3
Mafa-A1*077:01:02	H	I	3
Mafa-A1*078:01:01	H	S	11
Mafa-A1*085:02	C, V	A	1
Mafa-A1*086:03	H	?	10
Mafa-A1*089:06	V	V	3
Mafa-A1*092:01	C	A, V	1,3,5
Mafa-A1*092:03	H	U	8
Mafa-A1*092:04	C	A	6	Mamu-A1*092:03 (KF297355 – 1098bp)
Mafa-A1*124:01	C	S	9
Mafa-A1*130:01	V	A	6	Mamu-A1*130:01 (HG813262 – 1098bp)
Mafa-A2*01:01	V	V	1,5	Mamu-A2*01:03 (GQ902066 – 1098bp)
Mafa-A2*05:06:02	C, H	Z	12	Mamu-A2*05:04:03 (LN899643 – 1098bp); Mane-A2*05:19:01 (LN852049 – 1098bp)
Mafa-A2*05:22	C	Z	12
Mafa-A2*05:25:01	C, H, V	S, Z	12,13	Mamu-A2*05:15:04 (AB444914 – 657bp)
Mafa-A2*05:31	C, H	Z	12
Mafa-A2*05:40	C, V	?	10	Mamu-A2*05:01 (U41831 – 735bp)
Mafa-A2*05:42	H	?	10
Mafa-A2*05:45	C	?	10
Mafa-A2*05:46	V	M	10,14	Mamu-A2*05:21 (AM295935 – 1065bp)
Mafa-A2*05:50	C	P	15,16	Mamu-A2*05:36 (LN899652 – 1098bp)
Mafa-A2*05:51	V	?	10
Mafa-A2*05:55	C, V	A	6	Mamu-A2*05:03:02 (LN899641 – 1101bp); Mane-A2*05:14 (EF394348 – 822bp)
Mafa-A2*05:57:01	C, H, V	?	10	Mamu-A2*05:10 (LN899644 – 1098bp); Mane-A2*05:25 (LN870205 – 1098bp)
Mafa-A3*13:02	C, V	V	2	Mamu-A3*13:03 (AF157401 – 1098bp); Mane-A3*13:01:01 (EF010521,LN852052 – 1098bp)
Mafa-A3*13:07	C, V	V	1,5,10
Mafa-A3*13:14N	C	?	10	Mamu-A4*03:02 (EU429614 – 822bp)
Mafa-A3*13:15	C	S	9
Mafa-A4*01:02	C	A, P	2,17
Mafa-A4*01:08	C, V	?	10
Mafa-A4*14:01	C, H	I, U	4,9	Mamu-A4*14:04:02 (KC205311 – 692bp); Mane-A4*14:01:03 (LN875417 – 1011bp)
Mafa-A4*14:03	C, H, V	P, V	1,5,10,16	Mamu-A4*14:03:01 (EF580138 – 1101bp)
Mafa-A4*14:04	H	?	10
Mafa-A4*14:13	V	A	6
Mafa-A4*14:16	V	-	-	Mamu-A4*14:14 (HG813264 – 1068bp)
Mafa-A5*30:06	C	A	6
Mafa-B*001:01:01	C, H, V	A, V	2,6,18	Mamu-B*001:01:01 (U42837 – 1080bp)
Mafa-B*002:04	C, V	V	5,18	Mamu-B*002:03 (LT546138 – 1089bp)
Mafa-B*007:01:01	C, V	A, P, V	2,5,9,10,15,16,18,19	Mamu-B*007:03 (EF580149,EU682528 – 1089bp)
Mafa-B*007:01:05	C, V	V	5,18
Mafa-B*007:05	C, V	V	18
Mafa-B*007:08	C, V	-	-	Mamu-B*007:04:01 (GQ902078 – 1089bp); Mane-B*007:03 (LN852069 – 1089bp)
Mafa-B*008:01	V	V	18	Mamu-B*008:01 (U41830 – 1089bp)
Mafa-B*010:01	C	S, U	8,20	Mamu-B*010:01 (EU682525 – 1089bp)
Mafa-B*011:01	H	I, M, U	2,7,8,14,15
Mafa-B*013:03	C, V	?	10
Mafa-B*013:06	C	V	5,10,18
Mafa-B*013:09	C, V	A, V	6,10,18
Mafa-B*013:10	V	A, V	5,6,18
Mafa-B*013:11	C	U	8
Mafa-B*013:18	C	-	-	Mamu-B*013:01 (GU057843 – 1089bp); Mane-B*013:02 (LN852070 – 1089bp)
Mafa-B*014:01	C, V	U	8	Mamu-B*014:01 (KF791281 – 1089bp); Mane-B*014:02:02N (LN875427 – 1005bp)
Mafa-B*015:01	C, H	V	2,5
Mafa-B*015:05	C	-	-	Mamu-B*015:02 (AM902542 – 1056bp)
Mafa-B*016:01	H	I	7
Mafa-B*016:02	C	-	-	Mamu-B*016:02 (AB477395 – 1083bp)
Mafa-B*017:02	C	A, V	6,18
Mafa-B*018:01:01	C	A, I, V	2,6,9	Mamu-B*018:01 (AM902534 – 1056bp)
Mafa-B*019:03	H	M	14,15,21
Mafa-B*021:02	C, V	A	6	Mamu-B*021:02 (AM902536 – 1053bp)
Mafa-B*025:02	V	-	-	Mamu-B*025:01:01 (AM902531,KF855167 – 1086bp)
Mafa-B*028:02	C, V	A, V	2,6	Mamu-B*028:02:01 (AM902532 – 1056bp)
Mafa-B*028:03	V	P, U, V	2,8,16,18
Mafa-B*028:04	C, H	I, V	5,7,18
Mafa-B*030:01:01	C, H, V	A, V	2,18	Mamu-B*030:03:02 (AM902546 – 1056bp)
Mafa-B*030:01:05	C, H	-	-	Mamu-B*030:03:03 (HG813266,LN899678 – 1089bp)
Mafa-B*030:02	C	A, S, V	2,9,18
Mafa-B*030:03:01	C, H	A, V	2,6,18
Mafa-B*030:03:02	C, V	V	10,18	Mamu-B*030:02 (EU589241 – 1089bp)
Mafa-B*030:04	C	?	10
Mafa-B*030:15	C, V	A	6
Mafa-B*030:17	C, H, V	A	6,10
Mafa-B*031:03	C, H	-	-	Mane-B*031:01 (LN852085 – 1095bp)
Mafa-B*034:01	C	V	2	Mamu-B*034:05 (LM608037 – 687bp)
Mafa-B*034:03	C	V	5,18
Mafa-B*034:04	V	A	6
Mafa-B*036:01:02	C	M, U, V	8,14,18,21	Mamu-B*036:01:01 (AJ556886,KF855170 – 1080bp)
Mafa-B*037:01	C	M, U, V	2,5,8,14,18	Mamu-B*037:01 (GU080239 – 1089bp)
Mafa-B*038:01	C, H	A	6
Mafa-B*039:01	C, V	A, V	5,6,18	Mamu-B*039:01 (EF580146 – 1089bp)
Mafa-B*041:02	C, H, V	A, P	6,16	Mamu-B*041:01 (LN899682 – 1089bp)
Mafa-B*041:03	V	A	6
Mafa-B*043:01	C	S	9	Mamu-B*043:01 (EF580147 – 1089bp)
Mafa-B*044:01:01	C, H, V	A, U	5,6,8	Mamu-B*044:03 (KF791284 – 1080bp)
Mafa-B*044:04	H	M	14
Mafa-B*047:03	H	-	-	Mamu-B*047:04 (HG813268 – 1089bp)
Mafa-B*050:05	C, H	V	5,18
Mafa-B*050:08	C, H	P	16	Mamu-B*050:02 (LN899691 – 1080bp)
Mafa-B*056:01	C, V	A, I, U, V	2,6,8,9,18	Mamu-B*056:01 (GQ902079 – 1089bp)
Mafa-B*056:04	C	A	6
Mafa-B*056:05:01	C	A, V	5,6,18
Mafa-B*056:05:02	H	-	-	Mane-B*056:01 (FJ875237 – 1089bp)
Mafa-B*060:01	C, H, V	I, M	7,14	Mane-B*060:02 (LN875444 – 1080bp)
Mafa-B*060:04	C, V	I, M, V	9,14,18	Mamu-B*060:07 (LN899701 – 1080bp)
Mafa-B*060:12	V	-	-	Mamu-B*060:12 (LN899744 – 1080bp)
Mafa-B*061:03:01	C	A	6
Mafa-B*061:04:01	C	A	6	Mamu-B*061:02 (HG813270 – 1080bp)
Mafa-B*064:05	H	-	-	Mamu-B*064:01 (AJ556908 – 1053bp); Mane-B*064:02 (LN875446 – 1077bp)
Mafa-B*065:02	C, H	V	5,18	Mamu-B*065:03:03 (LT623026 – 1089bp)
Mafa-B*065:04	C, H, V	-	-	Mamu-B*065:02 (EF580163 – 1089bp)
Mafa-B*065:05	H	-	-	Mane-B*065:01:01 (LN875447 – 1089bp)
Mafa-B*066:01	V	A	6	Mamu-B*066:01 (EU682526,GQ902076 – 1089bp)
Mafa-B*067:01	C	-	-	Mamu-B*067:01:01 (KF791287 – 1089bp)
Mafa-B*068:02	C, H, V	U	8
Mafa-B*068:04	C, V	A, S	6,9	Mamu-B*068:04 (HG813271 – 1089bp)
Mafa-B*068:06:01	C, V	A	6	Mamu-B*068:02:01 (EF362453 – 1089bp); Mane-B*068:02:03 (LN852099 – 1089bp)
Mafa-B*068:07:01	V	A	6	Mamu-B*068:01:01 (KF791288 – 1089bp)
Mafa-B*069:03	C, H	V	18
Mafa-B*069:04	C, V	V	5,18
Mafa-B*069:05	C, H	-	-	Mamu-B*069:01 (EF580148 – 1089bp)
Mafa-B*069:06	C	-	-	Mamu-B*069:04 (EU682519 – 1089bp)
Mafa-B*069:07	C	?	5	Mamu-B*069:02 (EF580158 – 1089bp)
Mafa-B*070:04	C, V	A	6	Mamu-B*070:02 (AM902575,KF855195 – 1080bp)
Mafa-B*072:02	H	M	14
Mafa-B*073:02	C, H	A	5,6	Mamu-B*073:01 (AM902578,KF855199 – 1089bp)
Mafa-B*074:02	C, V	S	9	Mamu-B*074:03 (KF297364 – 1080bp)
Mafa-B*075:01	H	I, M, U	2,7,8,14,15
Mafa-B*077:01	V	U	8	Mamu-B*077:02 (AM902580 – 1056bp)
Mafa-B*079:01:01	H	I, M	14,22
Mafa-B*079:02:01	C, H, V	?	10	Mamu-B*079:01:01 (EF611165 – 1080bp); Mane-B*079:01:01 (GQ153468 – 1080bp)
Mafa-B*081:01	C, V	I, V	5,9,18
Mafa-B*082:03	C, H, V	-	-	Mane-B*082:02 (FJ875253 – 1012bp)
Mafa-B*083:02	C	A	6,10	Mamu-B*083:02 (AB542052 – 649bp)
Mafa-B*085:01	C, H	A, P, V	2,5,6,16,18
Mafa-B*088:04	C, V	-	-	Mamu-B*088:01:02 (LT546135 – 1089bp)
Mafa-B*089:01:02	C, V	I, P, S, V	5,9,10,16,17,18,19	Mamu-B*089:01:02 (GQ131743 – 822bp); Mane-B*089:03 (LN852118 – 1012bp)
Mafa-B*091:03	C	?	5
Mafa-B*093:02	C, V	V	5,18
Mafa-B*095:04	C	-	-	Mamu-B*095:02 (GQ131733 – 822bp)
Mafa-B*098:04	C, V	M	14	Mamu-B*098:06:02 (LT623046 – 1089bp)
Mafa-B*098:12	C, V	-	-	Mamu-B*098:01:01 (FN396415,GQ153380 – 1089bp)
Mafa-B*101:02	C, V	V	5,18
Mafa-B*104:01:02	C, H, V	U, V	5,8,18	Mane-B*104:02 (FJ875231 – 1089bp)
Mafa-B*105:01	H	V	5,18	Mamu-B*105:01 (AB540184 – 649bp); Mane-B*105:01 (LN870231 – 1089bp)
Mafa-B*110:01:01	C, V	V	5,18
Mafa-B*124:01:01	H	I	7
Mafa-B*124:01:03	C	A	6	Mamu-B*124:01 (LM608082 – 693bp); Mane-B*124:01 (AY204716,LN852144 – 1080bp)
Mafa-B*137:03	C, V	I, P, U, V	5,7,8,9,16,17,18,19
Mafa-B*139:02	V	U	8	Mamu-B*139:01 (LN899733 – 1086bp)
Mafa-B*144:01	C, V	V	2,5,18
Mafa-B*145:01	C, V	V	2,5,18
Mafa-B*146:01:01	C	V	2	Mamu-B*146:01 (GQ153418 – 692bp)
Mafa-B*149:01	C, V	M	14,21
Mafa-B*149:02	C	I	10,23
Mafa-B*150:02	H	U	8
Mafa-B*155:01	V	I, U	5,7,8	Mamu-B*155:01 (LM608079 – 687bp)
Mafa-B*156:01	H	I	7,10	Mamu-B*156:01 (AB540192 – 649bp)
Mafa-B*164:02	H	M, P	14,16,24
Mafa-B*184:01:01	V	?	10
Mafa-I*01:09	V	I, S, U	8,9,20	Mamu-I*01:29 (KF855226 – 1089bp); Mane-I*01:27 (LN875474 – 1089bp)
Mafa-I*01:11:02	C, H, V	-	-	Mamu-I*01:09:04 (KF855225 – 692bp)
Mafa-I*01:18:01	C, H, V	U	8
Mafa-I*01:22	H	M	14,25

Summary of geographical origins in which the 181 previously described cynomolgus macaque MHC class I transcripts were observed, and the 100 cynomolgus macaque sequences with 100% identity to transcripts observed in rhesus (Mamu) and/or pig-tailed (Mane) macaques. Cynomolgus macaque geographical origin abbreviations – A = Chinese breeding facility, C = Cambodia, H = Cambodia/Indonesia hybrid origins, I = Indonesia, M = Mauritius, P = Philippines, S = Malaysia, U = Utrecht breeding colony (Indonesia/Malaysia hybrid origins), V = Vietnam, Z = University of New Mexico study (Vietnam or Indonesia origins, not specified per sequence), ? = no origin information available for that transcript, - = sequence novel to this study (no previously described geographical origins). References for geographical origin information – 1 = Zhuo et al. unpublished sequence retrieved from GenBank, 2 = Krebs et al. 2005, 3 = Kita et al. 2009, 4 = Otting et al. 2007, 5 = Wang et al. unpublished sequence retrieved from GenBank, 6 = Westbrook et al. 2015, 7 = Pendley et al. 2008, 8 = Otting et al. 2009, 9 = Saito et al. 2012, 10 = Wu et al. unpublished sequence retrieved from GenBank, 11 = Uda et al. 2004, 12 = Wu et al. 2008, 13 = Aarnink et al. 2011, 14 = Budde et al. 2010, 15 = Aarnink et al. unpublished sequence retrieved from GenBank, 16 = Shiina et al. 2015, 17 = Lawrence et al. 2012, 18 = Wang et al. 2011, 19 = Campbell et al. 2008, 20 = Uda et al. 2005, 21 = Wiseman et al. 2007, 22 = Mee et al. unpublished sequence retrieved from GenBank, 23 = Otting et al. unpublished sequence retrieved from GenBank, 24 = Wiseman et al. 2009, 25 = Urvater et al. unpublished sequence retrieved from GenBank. For cynomolgus macaque transcripts that are identical to sequences observed in rhesus and pig-tailed macaques, the Mamu and/or Mane names are provided along with accession numbers for longest example of each sequence and the known length in bp

Similarly, 100 of the 311 transcripts identified in this study were identical to officially named sequences contained in the IPD databases for rhesus macaques (Macaca mulatta; Mamu) and/or pig-tailed macaques (Macaca nemestrina; Mane) (Table 2). Twenty-nine of these transcripts do not have a full-length ortholog in the rhesus or pig-tailed macaque databases, so these may prove to be distinct alleles if they contain any nucleotide polymorphisms outside the currently described sequence. These shared transcripts likely represent MHC class I alleles present in a common ancestor between cynomolgus, rhesus, and/or pig-tailed macaques. Overall, with high-throughput next generation sequencing methodologies making large-cohort MHC class I analysis studies feasible, more allele sharing is likely to be identified both amongst different geographical origins of macaques and between macaque species.

We also examined distinct, full resolution haplotypes in the samples that were PacBio sequenced to compare to the previously determined lineage-level haplotypes from the full cohort. Fig. 1b shows the PacBio haplotype results for two representative trio sets of samples, and Supplemental Table 2 provides the complete list of full resolution haplotypes observed in the cohort. Haplotype names were retained from the lineage-level haplotype results, but sub-haplotypes (indicated with Roman numerals in the “Sub-Haplo” column of Supplemental Table 2) were assigned for any distinctions in specific transcripts. For instance, the lineage-level haplotype A001 has four (i-iv) distinct sub-haplotypes in the PacBio data, as they all vary from each other at the transcript level (A001i – A001iv carry Mafa-A1*001:01:02, Mafa-A1*001:01:03, Mafa-A1*001:02:01, and Mafa-A1*001:05, respectively). For the PacBio haplotypes, both major and minor specific transcripts were considered when assigning sub-haplotypes. As expected, results were concurrent with the lineage-level haplotypes overall, but the full-length allelic resolution provided by PacBio sequencing resulted in a significant expansion of the number of both Mafa-A and Mafa-B haplotypes. In total we identified 92 and 101 distinct full-resolution Mafa-A and Mafa-B haplotypes, respectively, in the 72 PacBio-sequenced samples. As with the lineage-level haplotypes, more than 90% of the Mafa-A and Mafa-B PacBio haplotypes were only observed in individuals from a single origin - 26 Mafa-A and 29 Mafa-B haplotypes were distinct to Vietnamese-origin samples, 43 Mafa-A and 46 Mafa-B haplotypes were only observed in Cambodian-origin samples, and 15 Mafa-A and 15 Mafa-B haplotypes were distinct to the hybrid Cambodian/Indonesian-origin samples (Supplemental Table 2).

It is unclear how many of these allelic distinctions make a functional biological difference in regards to immune responses. Although such confirmatory studies are beyond the scope of this report, we predict that specific nonsynonymous nucleotide variants between two closely related alleles may result in significant biological differences, particularly when the nucleotide differences alter the resulting amino acid within the peptide binding pocket. Therefore, full resolution genotyping provided by PacBio sequencing is particularly attractive for researchers looking for linkage between a specific immune response and specific MHC class I genotypes.

Overall, this study describes some of the first MHC class I Mafa-A and Mafa-B haplotypes in cynomolgus macaques from Chinese breeding facilities, including samples of reported Vietnamese, Cambodian, and mixed Cambodian/Indonesian origins. It also describes an improved PacBio long read sequencing approach to characterize full-length MHC class I transcripts without contig assembly, using only the highest quality reads. The transcripts identified here both increase the total number of known Mafa-A, Mafa-B, and Mafa-I sequences, and significantly improve the percentage of full-length transcripts in the nonhuman primate IPD database. Finally, the general PacBio-based method described here for long-amplicon sequencing can also be applied to other complex immune loci of macaques such as MHC class II, killer immunoglobulin-like receptors, and Fc gamma receptors, as well as those of other model organisms.