Rapid high-resolution MHC class I genotyping of Chinese rhesus macaques by capillary reference strand-mediated conformational analysis

Rhesus macaques (Macaca mulatta) provide well-established models for studying human disease pathogenesis and vaccine development. When challenged with infectious agents, macaques exhibit individual differences in susceptibility. An important determinant of these differences is the complement of major histocompatability complex (MHC) class I sequences expressed by each animal. Although previous studies have reported strong associations between MHC expression and disease outcome, a rapid, cost-effective method for high-resolution MHC genotyping in macaques is lacking. In this study, we adapted a modified heteroduplex assay, reference strand-mediated conformational analysis (RSCA) to an ABI 3130xl capillary electrophoresis genetic analyzer for macaque MHC class I genotyping. For validation, we investigated the concordance of RSCA genotyping for 14 MHC class I sequences in 12 Chinese rhesus macaques whose genotypes were established through complementary DNA cloning and sequencing of MHC class I sequences. We observed a concordance greater than 98% between RSCA and the cloning and sequencing data. Furthermore, RSCA confirmed the presence of MHC haplotype sharing between three macaques as predicted previously by microsatellite analysis. RSCA genotyping of an additional 25 Chinese rhesus macaques demonstrated that the frequency of these 14 MHC class I sequences ranged from 5% to 32%, with the Mamu-A1*2601 sequence being most common in this cohort. Capillary RSCA genotyping has the potential to enable researchers to rapidly evaluate MHC class I genotypes in rhesus macaques and associate specific MHC sequences with disease susceptibility.     

New generation sequencers as a tool for genotyping of highly polymorphic multilocus MHC system

Accurate genotyping of complex systems, such as the major histocompatibility complex (MHC) often requires simultaneous analysis of multiple co-amplifying loci. Here we explore the utility of the massively parallel 454 sequencing method as a universal tool for genotyping complex MHC systems in nonmodel vertebrates. The power of this approach stems from the use of tagged polymerase chain reaction (PCR) primers to identify individual amplicons which can be simultaneously sequenced to the arbitrarily chosen coverage. However, the error-prone sequencing technology poses considerable challenges as it may be difficult to discriminate between sequencing errors and true rare alleles; due to complex nature of artefacts and errors, efficient quality control is required. Nevertheless, our study demonstrates the parallel 454 sequencing can be an efficient genotyping platform for MHC and provides an alternative to classical genotyping methods. We introduced procedures to identify the threshold that can be used to reduce number of genotyping errors by eliminating most of artefactual alleles (AA) representing PCR or sequencing errors. Our procedures are based on two expectations: first, that AA should be relatively rare, both overall and on per-individual basis, and second, that most AA result from errors introduced to sequences of true alleles. In our data set, alleles with an average per-individual frequency below 3% most likely represented artefacts. This threshold will vary in other applications according to the complexity of the genotyped system. We strongly suggest direct assessment of genotyping error in every experiment by running a fraction of duplicates: individuals amplified in independent PCRs.     

Is MHC diversity a better marker for conservation than neutral genetic diversity? A case study of two contrasting dolphin populations

Genetic diversity is essential for populations to adapt to changing environments. Measures of genetic diversity are often based on selectively neutral markers, such as microsatellites. Genetic diversity to guide conservation management, however, is better reflected by adaptive markers, including genes of the major histocompatibility complex (MHC). Our aim was to assess MHC and neutral genetic diversity in two contrasting bottlenose dolphin (Tursiops aduncus) populations in Western Australia—one apparently viable population with high reproductive output (Shark Bay) and one with lower reproductive output that was forecast to decline (Bunbury). We assessed genetic variation in the two populations by sequencing the MHC class II DQB, which encompasses the functionally important peptide binding regions (PBR). Neutral genetic diversity was assessed by genotyping twenty‐three microsatellite loci. We confirmed that MHC is an adaptive marker in both populations. Overall, the Shark Bay population exhibited greater MHC diversity than the Bunbury population—for example, it displayed greater MHC nucleotide diversity. In contrast, the difference in microsatellite diversity between the two populations was comparatively low. Our findings are consistent with the hypothesis that viable populations typically display greater genetic diversity than less viable populations. The results also suggest that MHC variation is more closely associated with population viability than neutral genetic variation. Although the inferences from our findings are limited, because we only compared two populations, our results add to a growing number of studies that highlight the usefulness of MHC as a potentially suitable genetic marker for animal conservation. The Shark Bay population, which carries greater adaptive genetic diversity than the Bunbury population, is thus likely more robust to natural or human‐induced changes to the coastal ecosystem it inhabits.     

jMHC: software assistant for multilocus genotyping of gene families using next-generation amplicon sequencing

Genotyping of multilocus gene families, such as the major histocompatibility complex (MHC), may be challenging because of problems with assigning alleles to loci and copy number variation among individuals. Simultaneous amplification and genotyping of multiple loci may be necessary, and in such cases, next-generation deep amplicon sequencing offers a great promise as a genotyping method of choice. Here, we describe jMHC, a computer program developed for analysing and assisting in the visualization of deep amplicon sequencing data. Software operates on FASTA files; therefore, output from any sequencing technology may be used. jMHC was designed specifically for MHC studies but it may be useful for analysing amplicons derived from other multigene families or for genotyping other polymorphic systems. The program is written in Java with user-friendly graphical interface (GUI) and can be run on Microsoft Windows, Linux OS and Mac OS.     

Family‐assisted inference of the genetic architecture of major histocompatibility complex variation

With their direct link to individual fitness, genes of the major histocompatibility complex (MHC) are a popular system to study the evolution of adaptive genetic diversity. However, owing to the highly dynamic evolution of the MHC region, the isolation, characterization and genotyping of MHC genes remain a major challenge. While high‐throughput sequencing technologies now provide unprecedented resolution of the high allelic diversity observed at the MHC, in many species, it remains unclear (i) how alleles are distributed among MHC loci, (ii) whether MHC loci are linked or segregate independently and (iii) how much copy number variation (CNV) can be observed for MHC genes in natural populations. Here, we show that the study of allele segregation patterns within families can provide significant insights in this context. We sequenced two MHC class I (MHC‐I) loci in 1267 European barn owls (Tyto alba), including 590 offspring from 130 families using Illumina MiSeq technology. Coupled with a high per‐individual sequencing coverage (~3000×), the study of allele segregation patterns within families provided information on three aspects of the architecture of MHC‐I variation in barn owls: (i) extensive sharing of alleles among loci, (ii) strong linkage of MHC‐I loci indicating tandem architecture and (iii) the presence of CNV in the barn owl MHC‐I. We conclude that the additional information that can be gained from high‐coverage amplicon sequencing by investigating allele segregation patterns in families not only helps improving the accuracy of MHC genotyping, but also contributes towards enhanced analyses in the context of MHC evolutionary ecology.     

Ultra‐deep Illumina sequencing accurately identifies MHC class IIb alleles and provides evidence for copy number variation in the guppy (Poecilia reticulata)

We address the bioinformatic issue of accurately separating amplified genes of the major histocompatibility complex (MHC) from artefacts generated during high‐throughput sequencing workflows. We fit observed ultra‐deep sequencing depths (hundreds to thousands of sequences per amplicon) of allelic variants to expectations from genetic models of copy number variation (CNV). We provide a simple, accurate and repeatable method for genotyping multigene families, evaluating our method via analyses of 209 b of MHC class IIb exon 2 in guppies (Poecilia reticulata). Genotype repeatability for resequenced individuals (N = 49) was high (100%) within the same sequencing run. However, repeatability dropped to 83.7% between independent runs, either because of lower mean amplicon sequencing depth in the initial run or random PCR effects. This highlights the importance of fully independent replicates. Significant improvements in genotyping accuracy were made by greatly reducing type I genotyping error (i.e. accepting an artefact as a true allele), which may occur when using low‐depth allele validation thresholds used by previous methods. Only a small amount (4.9%) of type II error (i.e. rejecting a genuine allele as an artefact) was detected through fully independent sequencing runs. We observed 1–6 alleles per individual, and evidence of sharing of alleles across loci. Variation in the total number of MHC class II loci among individuals, both among and within populations was also observed, and some genotypes appeared to be partially hemizygous; total allelic dosage added up to an odd number of allelic copies. Collectively, observations provide evidence of MHC CNV and its complex basis in natural populations.     

Critical review of NGS analyses for de novo genotyping multigene families

The genotyping of highly polymorphic multigene families across many individuals used to be a particularly challenging task because of methodological limitations associated with traditional approaches. Next‐generation sequencing (NGS) can overcome most of these limitations, and it is increasingly being applied in population genetic studies of multigene families. Here, we critically review NGS bioinformatic approaches that have been used to genotype the major histocompatibility complex (MHC) immune genes, and we discuss how the significant advances made in this field are applicable to population genetic studies of gene families. Increasingly, approaches are introduced that apply thresholds of sequencing depth and sequence similarity to separate alleles from methodological artefacts. We explain why these approaches are particularly sensitive to methodological biases by violating fundamental genotyping assumptions. An alternative strategy that utilizes ultra‐deep sequencing (hundreds to thousands of sequences per amplicon) to reconstruct genotypes and applies statistical methods on the sequencing depth to separate alleles from artefacts appears to be more robust. Importantly, the ‘degree of change’ (DOC) method avoids using arbitrary cut‐off thresholds by looking for statistical boundaries between the sequencing depth for alleles and artefacts, and hence, it is entirely repeatable across studies. Although the advances made in generating NGS data are still far ahead of our ability to perform reliable processing, analysis and interpretation, the community is developing statistically rigorous protocols that will allow us to address novel questions in evolution, ecology and genetics of multigene families. Future developments in third‐generation single molecule sequencing may potentially help overcome problems that still persist in de novo multigene amplicon genotyping when using current second‐generation sequencing approaches.     

Influence of the MHC genotype on the progression of experimental SIV infection in the Mauritian cynomolgus macaque

Experimental infection of Mauritian cynomolgus macaques by simian immunodeficiency virus is a representative model of HIV infection, currently in favour for evaluating the efficacy of new preventive or curative treatments. Extensive studies of major histocompatibility complex (MHC) polymorphism by microsatellites revealed seven haplotypes (H1–H7). We present statistical evidence of the influence of MHC polymorphism on the set-point plasma viral load (PVL). Our analysis was based on the study of 45 Mauritian cynomolgus macaques inoculated by intravenous or intrarectal injection of a 50 AID50 dose of the SIVmac251 virus. The animals received no treatment before or after the inoculation. MHC polymorphism was investigated by means of 20 microsatellites distributed across the MHC and by DRB genotyping using the DGGE sequencing method. Statistical analysis with Unphased software revealed that two markers located in the class IB region significantly influenced the Log PVL and that three class IB haplotypes were significantly associated with lower (H2 or H6) or higher (H4) set-point Log PVL values. Although the impact of MHC on Log PVL was found to be low (around one Log10), it is important to dispose of animals paired for their MHC genotypes, each animal tested for a given treatment and its untreated control, to minimize the influence of the MHC and clearly reveal the effect of the treatment.     

Testing genotyping strategies for ultra‐deep sequencing of a co‐amplifying gene family: MHC class I in a passerine bird

Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co‐amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra‐deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500–20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within‐method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co‐amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co‐amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage.     

Evaluation of two approaches to genotyping major histocompatibility complex class I in a passerine-CE-SSCP and 454 pyrosequencing

Genes of the highly dynamic major histocompatibility complex (MHC) are directly linked to individual fitness and are of high interest in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high levels of polymorphism in the MHC region, making genotyping of MHC a challenging task. Here, we compare the performance of two methods for MHC class I genotyping in a passerine with highly duplicated MHC class I genes: capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis and 454 GS FLX Titanium pyrosequencing. According to our findings, the number of MHC variants (called alleles for simplicity) detected by CE-SSCP is significantly lower than detected by 454. To resolve discrepancies between the two methods, we cloned and Sanger sequenced a MHC class I amplicon for an individual with high number of alleles. We found a perfect congruence between cloning/Sanger sequencing results and 454. Thus, in case of multi-locus amplification, CE-SSCP considerably underestimates individual MHC diversity. However, numbers of alleles detected by both methods are significantly correlated, although the correlation is weak (r = 0.32). Thus, in systems with highly duplicated MHC, 454 provides more reliable information on individual diversity than CE-SSCP.     

Stepwise Threshold Clustering: A New Method for Genotyping MHC Loci Using Next-Generation Sequencing Technology

Genes of the vertebrate major histocompatibility complex (MHC) are of great interest to biologists because of their important role in immunity and disease, and their extremely high levels of genetic diversity. Next generation sequencing (NGS) technologies are quickly becoming the method of choice for high-throughput genotyping of multi-locus templates like MHC in non-model organisms.Previous approaches to genotyping MHC genes using NGS technologies suffer from two problems:1) a “gray zone” where low frequency alleles and high frequency artifacts can be difficult to disentangle and 2) a similar sequence problem, where very similar alleles can be difficult to distinguish as two distinct alleles. Here were present a new method for genotyping MHC loci – Stepwise Threshold Clustering (STC) – that addresses these problems by taking full advantage of the increase in sequence data provided by NGS technologies. Unlike previous approaches for genotyping MHC with NGS data that attempt to classify individual sequences as alleles or artifacts, STC uses a quasi-Dirichlet clustering algorithm to cluster similar sequences at increasing levels of sequence similarity. By applying frequency and similarity based criteria to clusters rather than individual sequences, STC is able to successfully identify clusters of sequences that correspond to individual or similar alleles present in the genomes of individual samples. Furthermore, STC does not require duplicate runs of all samples, increasing the number of samples that can be genotyped in a given project. We show how the STC method works using a single sample library. We then apply STC to 295 threespine stickleback (Gasterosteus aculeatus) samples from four populations and show that neighboring populations differ significantly in MHC allele pools. We show that STC is a reliable, accurate, efficient, and flexible method for genotyping MHC that will be of use to biologists interested in a variety of downstream applications.     

Large-scale MHC class II genotyping of a wild lemur population by next generation sequencing

The critical role of major histocompatibility complex (MHC) genes in disease resistance, along with their putative function in sexual selection, reproduction and chemical ecology, make them an important genetic system in evolutionary ecology. Studying selective pressures acting on MHC genes in the wild nevertheless requires population-wide genotyping, which has long been challenging because of their extensive polymorphism. Here, we report on large-scale genotyping of the MHC class II loci of the grey mouse lemur (Microcebus murinus) from a wild population in western Madagascar. The second exons from MHC-DRB and -DQB of 772 and 672 individuals were sequenced, respectively, using a 454 sequencing platform, generating more than 800,000 reads. Sequence analysis, through a stepwise variant validation procedure, allowed reliable typing of more than 600 individuals. The quality of our genotyping was evaluated through three independent methods, namely genotyping the same individuals by both cloning and 454 sequencing, running duplicates, and comparing parent–offspring dyads; each displaying very high accuracy. A total of 61 (including 20 new) and 60 (including 53 new) alleles were detected at DRB and DQB genes, respectively. Both loci were non-duplicated, in tight linkage disequilibrium and in Hardy–Weinberg equilibrium, despite the fact that sequence analysis revealed clear evidence of historical selection. Our results highlight the potential of 454 sequencing technology in attempts to investigate patterns of selection shaping MHC variation in contemporary populations. The power of this approach will nevertheless be conditional upon strict quality control of the genotyping data.     

Methods for MHC genotyping in non-model vertebrates

Genes of the major histocompatibility complex (MHC) are considered a paradigm of adaptive evolution at the molecular level and as such are frequently investigated by evolutionary biologists and ecologists. Accurate genotyping is essential for understanding of the role that MHC variation plays in natural populations, but may be extremely challenging. Here, I discuss the DNA-based methods currently used for genotyping MHC in non-model vertebrates, as well as techniques likely to find widespread use in the future. I also highlight the aspects of MHC structure that are relevant for genotyping, and detail the challenges posed by the complex genomic organization and high sequence variation of MHC loci. Special emphasis is placed on designing appropriate PCR primers, accounting for artefacts and the problem of genotyping alleles from multiple, co-amplifying loci, a strategy which is frequently necessary due to the structure of the MHC. The suitability of typing techniques is compared in various research situations, strategies for efficient genotyping are discussed and areas of likely progress in future are identified. This review addresses the well established typing methods such as the Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Reference Strand Conformational Analysis (RSCA) and cloning of PCR products. In addition, it includes the intriguing possibility of direct amplicon sequencing followed by the computational inference of alleles and also next generation sequencing (NGS) technologies; the latter technique may, in the future, find widespread use in typing complex multilocus MHC systems.     

Solutions for PCR,cloning and sequencing errors in population genetic analysis

PCR and sequencing artefacts can seriously bias population genetic analyses, particularly of populations with low genetic variation such as endangered vertebrate populations. Here, we estimate the error rates, discuss their population genetics implications, and propose a simple detection method that helps to reduce the risk of accepting such errors. We study the major histocompatibility complex (MHC) class IIB of guppies, Poecilia reticulata and find that PCR base misincorporations inflate the apparent sequence diversity. When analysing neutral genes, such bias can inflate estimates of effective population size. Previously suggested protocols for identifying genuine alleles are unlikely to exclude all sequencing errors, or they ignore genuine sequence diversity. We present a novel and statistically robust method that reduces the likelihood of accepting PCR artefacts as genuine alleles, and which minimises the necessity of repeated genotyping. Our method identifies sequences that are unlikely to be a PCR artefact, and which need to be independently confirmed through additional PCR of the same template DNA. The proposed methods are recommended particularly for population genetic studies that involve multi-template DNA and in studies on genes with low genetic diversity.     

MHC polymorphism in Caribbean African green monkeys

African green monkeys (AGM) are among the most widely used nonhuman primate models used in various fields of medical research. One species of AGM that originated from West Africa, Chlorocebus sabaeus, was introduced three centuries ago in the Caribbean islands. We present here a systematic study of the major histocompatibility complex (MHC) polymorphism of Caribbean AGM which is currently frequently used as an animal model. We studied 54 animals originated from Barbados (N?=?25) or Saint Kitts (N?=?29). The MHC polymorphism was characterized by means of 17 MHC microsatellites spread across MHC and DRB genotyping by DGGE sequencing. We defined nine frequent MHC haplotypes of which two were found in the two insular populations suggesting either past exchanges between the two populations or a common origin of the founders of the two populations. By the analysis of a previously described EST library, we characterized 38 MHC cDNA sequences (17 class I and 21 class II). In conclusion, we characterized for the first time the MHC polymorphism of Barbados and Saint Kitts AGM. We found a restricted polymorphism due to a founding effect, which is responsible for a strong bottleneck. The poorness of MHC polymorphism observed in the Caribbean AGM populations is similar to that observed in the Mauritian cynomolgus macaque population.