High-throughput Single Nucleotide Polymorphism Research Articles

Estimating age of admixture in a cattle population based on SNP chip data

The aim of this study was to predict individual age of admixture in the crossbred Swiss Fleckvieh population. We checked how well the method is dealing with recent admixture with high throughput single nucleotide polymorphism data from the bovine 50K SNP Chip. A total of 101 Red Holstein, 91 Simmental, and 308 crossed animals were available for analysis. Age of admixture was derived from the complete pedigree and molecular markers. The method applied (using SABER software) based on Markov-hidden Markov model was able to derive age of admixture similar to estimates of pedigree data, however the values were often overestimated. Of 21 investigated cases, results from SNP data reflected paternal and maternal age of admixture well for 9 cases but provided results out of range for the other 12 cases. Alternative methods based on breed-specific haplotype blocks need to be evaluated in the future.

High-Throughput SNP Discovery and Genotyping for Constructing a Saturated Linkage Map of Chickpea (Cicer arietinum L.)

The present study reports the large-scale discovery of genome-wide single-nucleotide polymorphisms (SNPs) in chickpea, identified mainly through the next generation sequencing of two genotypes, i.e. Cicer arietinum ICC4958 and its wild progenitor C. reticulatum PI489777, parents of an inter-specific reference mapping population of chickpea. Development and validation of a high-throughput SNP genotyping assay based on Illumina's GoldenGate Genotyping Technology and its application in building a high-resolution genetic linkage map of chickpea is described for the first time. In this study, 1022 SNPs were identified, of which 768 high-confidence SNPs were selected for designing the custom Oligo Pool All (CpOPA-I) for genotyping. Of these, 697 SNPs could be successfully used for genotyping, demonstrating a high success rate of 90.75%. Genotyping data of the 697 SNPs were compiled along with those of 368 co-dominant markers mapped in an earlier study, and a saturated genetic linkage map of chickpea was constructed. One thousand and sixty-three markers were mapped onto eight linkage groups spanning 1808.7 cM (centiMorgans) with an average inter-marker distance of 1.70 cM, thereby representing one of the most advanced maps of chickpea. The map was used for the synteny analysis of chickpea, which revealed a higher degree of synteny with the phylogenetically close Medicago than with soybean. The first set of validated SNPs and map resources developed in this study will not only facilitate QTL mapping, genome-wide association analysis and comparative mapping in legumes but also help anchor scaffolds arising out of the whole-genome sequencing of chickpea.

The Effect of Input DNA Copy Number on Genotype Call and Characterising SNP Markers in the Humpback Whale Genome Using a Nanofluidic Array

Recent advances in nanofluidic technologies have enabled the use of Integrated Fluidic Circuits (IFCs) for high-throughput Single Nucleotide Polymorphism (SNP) genotyping (GT). In this study, we implemented and validated a relatively low cost nanofluidic system for SNP-GT with and without Specific Target Amplification (STA). As proof of principle, we first validated the effect of input DNA copy number on genotype call rate using well characterised, digital PCR (dPCR) quantified human genomic DNA samples and then implemented the validated method to genotype 45 SNPs in the humpback whale, Megaptera novaeangliae, nuclear genome. When STA was not incorporated, for a homozygous human DNA sample, reaction chambers containing, on average 9 to 97 copies, showed 100% call rate and accuracy. Below 9 copies, the call rate decreased, and at one copy it was 40%. For a heterozygous human DNA sample, the call rate decreased from 100% to 21% when predicted copies per reaction chamber decreased from 38 copies to one copy. The tightness of genotype clusters on a scatter plot also decreased. In contrast, when the same samples were subjected to STA prior to genotyping a call rate and a call accuracy of 100% were achieved. Our results demonstrate that low input DNA copy number affects the quality of data generated, in particular for a heterozygous sample. Similar to human genomic DNA, a call rate and a call accuracy of 100% was achieved with whale genomic DNA samples following multiplex STA using either 15 or 45 SNP-GT assays. These calls were 100% concordant with their true genotypes determined by an independent method, suggesting that the nanofluidic system is a reliable platform for executing call rates with high accuracy and concordance in genomic sequences derived from biological tissue.

Recent Patents on High-Throughput Single Nucleotide Polymorphism (SNP) Genotyping Methods

Single nucleotide polymorphisms (SNPs) are single-base inheritable variations in a given and defined genetic location that occur in at least 1% of the population. SNPs are useful markers for genetic association studies in disease susceptibility or adverse drug reactions, in evolutionary studies and forensic science. Given the potential impact of SNPs, the biotechnology industry has focused on the development of high-throughput methods for SNP genotyping. Many highthroughput SNP genotyping technologies are currently available and many others are being patented recently. Each offers a unique combination of scale, accuracy, throughput and cost. In this review, we described some of the most important recent SNP genotyping methods and also recent patents associated with it.

Single nucleotide polymorphisms in Cryptomeria japonica: their discovery and validation for genome mapping and diversity studies

In order to develop a large set of single-nucleotide polymorphisms (SNPs) in Cryptomeria japonica, for a wide range of applications, we adopted a systematic EST (expressed sequence tags) re-sequencing approach. We examined a group of four genotypes comprising parents of a mapping population as well as representatives of two main lines from natural populations. We sequenced 5,170 gene fragments, representing analysis of over 1.3 Mb of DNA sequences in C. japonica. This analysis leads to the discovery of 13,413 SNPs in 3,744 amplicons, with an average of one SNP for every 101.0 bp (one SNP for every 78.3 bp in introns and for every 106.7 bp in exon regions). Nucleotide diversity in C. japonica (π = 0.0045) was found to be similar to values recorded in highly polymorphic forest tree species such as pine. We also validated the use of the SNPs as molecular markers for genetic diversity studies using the high throughput SNP genotyping platform GoldenGate. From 1,536 candidate SNP sites tested, 1,164 (75.8 %) were confirmed to be polymorphic. We anticipate that the genome-wide SNP markers reported here will be useful for evaluating the species’ range-wide genetic structure and in marker-assisted selection used as part of the C. japonica tree improvement program.

High degree of single nucleotide polymorphisms in California Culex pipiens (Diptera: Culicidae) sensu lato.

Resolution of systematic relationships among members of the Culex pipiens (L.) complex has important implications for public health as well as for studies on the evolution of sibling species. Currently held views contend that in California considerable genetic introgression occurs between Cx. pipiens and Cx. quinquefasciatus Say, and as such, these taxa behave as if they are a single species. Development of high throughput SNP genotyping tools for the analysis of Cx. pipiens complex population structure is therefore desirable. As a first step toward this goal, we sequenced 12 gene fragments from specimens collected in Marin and Fresno counties. On average, we found a higher single nucleotide polymorphism (SNP) density than any other mosquito species reported thus far. Coding regions contained significantly higher GC content (median 54.7%) than noncoding regions (42.4%; Wilcoxon rank sum test, P = 5.29 x 10(-5)). Differences in SNP allele frequencies observed between mosquitoes from Marin and Fresno counties indicated significant genetic divergence and suggest that SNP markers will be useful for future detailed population genetic studies of this group. The high density of SNPs highlights the difficulty in identifying species within the complex and may be associated with the large degree of phenotypic variation observed in this group of mosquitoes.

Single-nucleotide polymorphisms for high-throughput genotyping of Anopheles arabiensis in East and southern Africa.

Anopheles arabiensis Patton is one of the principal vectors of malaria in sub-Saharan Africa, occupying a wide variety of ecological zones. This species is increasingly responsible for malaria transmission in Africa and is becoming the dominant vector species in some localities. Despite its growing importance, little is known about genetic polymorphisms in this species. Multiple sequences of various gene fragments from An. arabiensis isolates from Cameroon were obtained from GenBank. In total, 20 gene fragments containing single-nucleotide polymorphisms (SNPs) at moderate density were selected for direct sequencing from field collected specimens from Tanzania and Zambia. We obtained 301 SNPs in total from the 20 gene fragments, 60 of which were suitable for Illumina GoldenGate SNP genotyping. A greater number of SNPs (n = 185) was suitable for analysis using Sequenom iPLEX, an alternative high-throughput genotyping technology using mass spectrometry. An SNP was present every 59 (+/- 44.5) bases on average. Overall, An. arabiensis from Tanzania and Zambia are genetically closer (mean F(ST) = 0.075) than either is to populations in Cameroon (F(ST, TZ-CM) = 0.250, F(ST,ZA-CM) = 0.372). A fixed polymorphism between East/southern and Central Africa was identified on AGAP000574, a gene on the X chromosome. We have identified SNPs in natural populations of An. arabiensis. SNP densities in An. arabiensis were higher than Anopheles gambiae s.s., suggesting a greater challenge in the development of high-throughput SNP analysis for this species. The SNP markers provided in this study are suitable for a high-throughput genotyping analysis and can be used for population genetic studies and association mapping efforts.

High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping

BackgroundCucurbita pepo is a member of the Cucurbitaceae family, the second- most important horticultural family in terms of economic importance after Solanaceae. The "summer squash" types, including Zucchini and Scallop, rank among the highest-valued vegetables worldwide. There are few genomic tools available for this species.The first Cucurbita transcriptome, along with a large collection of Single Nucleotide Polymorphisms (SNP), was recently generated using massive sequencing. A set of 384 SNP was selected to generate an Illumina GoldenGate assay in order to construct the first SNP-based genetic map of Cucurbita and map quantitative trait loci (QTL).ResultsWe herein present the construction of the first SNP-based genetic map of Cucurbita pepo using a population derived from the cross of two varieties with contrasting phenotypes, representing the main cultivar groups of the species' two subspecies: Zucchini (subsp. pepo) × Scallop (subsp. ovifera). The mapping population was genotyped with 384 SNP, a set of selected EST-SNP identified in silico after massive sequencing of the transcriptomes of both parents, using the Illumina GoldenGate platform. The global success rate of the assay was higher than 85%. In total, 304 SNP were mapped, along with 11 SSR from a previous map, giving a map density of 5.56 cM/marker. This map was used to infer syntenic relationships between C. pepo and cucumber and to successfully map QTL that control plant, flowering and fruit traits that are of benefit to squash breeding. The QTL effects were validated in backcross populations.ConclusionOur results show that massive sequencing in different genotypes is an excellent tool for SNP discovery, and that the Illumina GoldenGate platform can be successfully applied to constructing genetic maps and performing QTL analysis in Cucurbita. This is the first SNP-based genetic map in the Cucurbita genus and is an invaluable new tool for biological research, especially considering that most of these markers are located in the coding regions of genes involved in different physiological processes. The platform will also be useful for future mapping and diversity studies, and will be essential in order to accelerate the process of breeding new and better-adapted squash varieties.

SNP Discovery and Development of a High-Density Genotyping Array for Sunflower

Recent advances in next-generation DNA sequencing technologies have made possible the development of high-throughput SNP genotyping platforms that allow for the simultaneous interrogation of thousands of single-nucleotide polymorphisms (SNPs). Such resources have the potential to facilitate the rapid development of high-density genetic maps, and to enable genome-wide association studies as well as molecular breeding approaches in a variety of taxa. Herein, we describe the development of a SNP genotyping resource for use in sunflower (Helianthus annuus L.). This work involved the development of a reference transcriptome assembly for sunflower, the discovery of thousands of high quality SNPs based on the generation and analysis of ca. 6 Gb of transcriptome re-sequencing data derived from multiple genotypes, the selection of 10,640 SNPs for inclusion in the genotyping array, and the use of the resulting array to screen a diverse panel of sunflower accessions as well as related wild species. The results of this work revealed a high frequency of polymorphic SNPs and relatively high level of cross-species transferability. Indeed, greater than 95% of successful SNP assays revealed polymorphism, and more than 90% of these assays could be successfully transferred to related wild species. Analysis of the polymorphism data revealed patterns of genetic differentiation that were largely congruent with the evolutionary history of sunflower, though the large number of markers allowed for finer resolution than has previously been possible.

From the Editors—Animal selection: The genomics revolution

The rapid improvements in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, ever-denser SNP arrays accompanied by reduced costs for genotyping and for sequencing, open the possibility of using genomic information in livestock selection. The industry is thus facing the new paradigm of “genomic selection,” in which genomic information may reduce costs and accelerate genetic gain by reducing generation intervals. Genetic markers, or SNP, are highly abundant in the genome, and highthroughput molecular technologies allow the inheritance of hundreds of thousands of such markers to be traced through generations for currently less than US $200 per animal. Even if the mutations themselves do not directly affect the phenotype, they can track for the variability of causal mutations in their vicinity. By summing the effects assigned to each SNP, we can create a numeric value (genomic breeding value) that allows the genetic potential of a breeding animal to be assessed. This is an enormously valuable tool for choosing breeding animals as parents of the next generation. In addition, genomic selection may allow the identifi cation of superior individuals for traits not currently considered in animal breeding plans because of technical diffi culties. For example, the fatty acid composition of dairy and beef food products, increased disease resistance (and thus increased animal welfare), and decreased methane emissions in cattle help to address the needs of consumers and society for sustainable and cost-effective food production. The animal breeding industry is currently adapting selection procedures in each species to include this innovative tool. This is causing a worldwide restructuring of the animal breeding industry. As an example in dairy cattle, large international consortia are being formed to develop joint precompetitive research and tools. For similar reasons, in the future, genomic selection might help close the gap between countries with greater and lesser production. The scope of this issue of Animal Frontiers is to provide a worldwide overview and various perspectives on intermediate-term scenarios resulting from the application of genomic tools in different livestock species. Genomic selection has already had a huge impact in genetic selectionrelated industries. Such industries have had the same structure for at least 3 decades. Subsequent to the research conducted by animal geneticists in the 1960s and 1970s, mainly by Charles Henderson and his group, animal selection remained the same for many years. Of course, new traits were included and new species were selected in addition to dairy cattle, but the industry structure generally remained the same. During these years, genetic selection also incorporated new computer technologies and animal reproduction discoveries to improve the identifi cation of superior animals. The genomic revolution created large changes in this structure. New actors appeared on the scene. In this issue of Animal Frontiers, relevant technicians and scientists were invited to focus on the impact of the genomic revolution on the genetics industry and to forecast future developments.

Comparison of a multiplexed MassARRAY system with real-time allele-specific PCR technology for genotyping of methicillin-resistant Staphylococcus aureus

The Sequenom MassARRAY iPLEX single-nucleotide polymorphism (SNP) typing platform uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with single-base extension PCR for high-throughput multiplex SNP detection. In this study, we investigated the use of iPLEX MassARRAY technology for methicillin-resistant Staphylococcus aureus (MRSA) genotyping. A 16-plex MassARRAY iPLEX GOLD assay (MRSA-iPLEX) was developed that targets a set of informative SNPs and binary genes for MRSA characterization. The method was evaluated with 147 MRSA isolates, and the results were compared with those of an established SYBR Green-based real-time PCR system utilizing the same SNP–binary markers. A total of 2352 markers belonging to 44 SNP–binary profiles were analysed by both real-time PCR and MRSA-iPLEX. With real-time PCR as the reference standard, MRSA-iPLEX correctly assigned 2298 of the 2352 (97.7%) markers. Sequence variation in the MRSA-iPLEX primer targets accounted for the majority of MRSA-iPLEX erroneous results, highlighting the importance of primer target selection. MRSA-iPLEX provided optimal throughput for MRSA genotyping, and was, on a reagent basis, more cost-effective than the real-time PCR methods. The 16-plex MRSA-iPLEX is a suitable alternative to SYBR Green-based real-time PCR typing of major sequence types and clonal complexes of MRSA.

High-throughput single nucleotide polymorphism genotyping for breeding applications in rice using the BeadXpress platform

Multiplexed single nucleotide polymorphism (SNP) markers have the potential to increase the speed and cost-effectiveness of genotyping, provided that an optimal SNP density is used for each application. To test the efficiency of multiplexed SNP genotyping for diversity, mapping and breeding applications in rice (Oryza sativa L.), we designed seven GoldenGate VeraCode oligo pool assay (OPA) sets for the Illumina BeadXpress Reader. Validated markers from existing 1536 Illumina SNPs and 44 K Affymetrix SNP chips developed at Cornell University were used to select subsets of informative SNPs for different germplasm groups with even distribution across the genome. A 96-plex OPA was developed for quality control purposes and for assigning a sample into one of the five O. sativa population subgroups. Six 384-plex OPAs were designed for genetic diversity analysis, DNA fingerprinting, and to have evenly-spaced polymorphic markers for quantitative trait locus (QTL) mapping and background selection for crosses between different germplasm pools in rice: Indica/Indica, Indica/Japonica, Japonica/Japonica, Indica/O. rufipogon, and Japonica/O. rufipogon. After testing on a diverse set of rice varieties, two of the SNP sets were re-designed by replacing poor-performing SNPs. Pilot studies were successfully performed for diversity analysis, QTL mapping, marker-assisted backcrossing, and developing specialized genetic stocks, demonstrating that 384-plex SNP genotyping on the BeadXpress platform is a robust and efficient method for marker genotyping in rice.

Malic enzyme gene polymorphism is associated with responsiveness in circulating parathyroid hormone after long-term calcium supplementation.

To identify genetic variations associated with parathyroid hormone (PTH) suppression after long-term calcium supplementation. For high throughput SNP screening, subjects consisted of 171 postmenopausal women without osteoporosis at the lumbar spine. A separate group of 19 premenpausal women were recruited for calcium absorption study. Postmenopausal women in the screening group were given 500 mg/day calcium supplementation. Bangkok, Thailand. Parathyroid hormone (PTH) and bone mineral density (BMD) were measured at baseline and 2 years after calcium supplementation. High throughput single-nucleotide polymorphism (SNP) screening was performed by comparing estimated allele frequencies derived from hybridization signal intensities of pooled DNA samples on Affymetrix's 10K SNP genotyping microarrays based responsiveness in PTH after calcium supplementation. Genotyping of SNP rs1112482 in malic enzyme gene (ME1) gene, a SNP among those with highest odds ratio of being related to PTH suppression after calcium, was performed in all postmenopausal subjects in the screening group and premenopausal women in the calcium absorption study group in which fractional calcium absorption was assessed by stable isotope dilution. Data were expressed as mean +/- SEM. PTH significantly decreased after 2 years of calcium supplementation (4.7 ± 1.9 vs. 4.4 ± 1.6 pmol/L, P < 0.01). There was a significant increase in lumbar spine BMD (1.03 ± 0.01 vs. 1.01 ± 0.01 g/cm2, P < 0.001) but not femoral neck BMD. In 108 subjects whose PTH levels decreased after calcium, the suppression of PTH was higher in those with at least one C allele in rs1112482 of ME1 gene (-26.3 ± 2.1 vs. -16.9 ± 1.4%, P < 0.001). Fractional calcium absorption also tends to the higher in subjects in the calcium absorption study group with at least one C allele (n = 6) compared to those without the C allele (n = 13) (58.0 ± 4.9 vs. 49.3 ± 2.8%, P = 0.054). Cytosolic malic enzyme 1 gene polymorphism is associated with the degree of suppression of parathyroid hormone after long-term calcium supplementation. The effect is probably mediated through an increase in intestinal calcium absorption.

From RNA-seq to large-scale genotyping - genomics resources for rye (Secale cereale L.)

BackgroundThe improvement of agricultural crops with regard to yield, resistance and environmental adaptation is a perpetual challenge for both breeding and research. Exploration of the genetic potential and implementation of genome-based breeding strategies for efficient rye (Secale cereale L.) cultivar improvement have been hampered by the lack of genome sequence information. To overcome this limitation we sequenced the transcriptomes of five winter rye inbred lines using Roche/454 GS FLX technology.ResultsMore than 2.5 million reads were assembled into 115,400 contigs representing a comprehensive rye expressed sequence tag (EST) resource. From sequence comparisons 5,234 single nucleotide polymorphisms (SNPs) were identified to develop the Rye5K high-throughput SNP genotyping array. Performance of the Rye5K SNP array was investigated by genotyping 59 rye inbred lines including the five lines used for sequencing, and five barley, three wheat, and two triticale accessions. A balanced distribution of allele frequencies ranging from 0.1 to 0.9 was observed. Residual heterozygosity of the rye inbred lines varied from 4.0 to 20.4% with higher average heterozygosity in the pollen compared to the seed parent pool.ConclusionsThe established sequence and molecular marker resources will improve and promote genetic and genomic research as well as genome-based breeding in rye.

Development of highly-multiplexed SNP arrays in maritime pine for multi-objective genetic applications

Single Nucleotide Polymorphisms (SNPs) are the most abundant form of genetic variation in the genome. In this poster, we reviewed what has been achieved in terms of highly-multiplexed SNP genotyping assay construction in maritime pine (Pinus pinaster Ait.), the main conifer used for commercial plantation in southwestern Europe. Seven custom SNP-assays (384 and 1536-plex), oriented towards broad applications, have been designed. We illustrated here the usefulness of this genotyping technology to address specific questions related to i/ genetic diversity and population structure analysis, ii/ linkage and QTL mapping and iii/ association mapping. With respect to genetic diversity and differentiation, a custom VeraCode assay for 384 SNPs mostly based on a larger array designed for linkage mapping (see below) allowed to obtain less blurred, albeit similar, breeding zone boundaries than a set of 12 nuSSRs screened on the same individuals. Levels of diversity were also more accurately estimated, showing clear differences among gene pools. Interestingly, a relatively small subset of SNPs would be enough to develop an application tool for origin certification, which could have a notable impact on current operational practices. In terms of linkage mapping, a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (in vitro SNPs) and from Sanger-derived Expressed Sequenced Tags (in silico SNPs) was established. Offspring from two mapping pedigrees were genotyped. A consensus map comprising 357 SNPs from 292 different loci was constructed and the analysis of sequence homology between mapped markers and their orthologs in a Pinus taeda linkage map, made it possible to align the 12 linkage groups of both species. Moreover, QTL detection for different traits is underway. In terms of association mapping, a custom GoldenGate assay with 384 SNPs was built and used to genotype 160 unrelated plus-trees from a half-sib experimental design for which breeding values (for height growth, circumference, stem straightness at 8 years, lignin content and extractives at 31 years) were available. Taking into account multiple testing, one single SNP in a gene encoding a putative fasciclin-like arabinogalactan protein was found to be associated with growth traits. We conclude that the VeraCode/GoldenGate assays can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This first generation of SNP-arrays has been recently upgraded to an Infinium-array (containing 10.5k SNPs) thanks to deep sequencing based on new generation sequencing technologies. The Infinium-array also includes SNPs from comparative orthologous sequences with other major conifer species, providing a wider collection of anchor points for comparative genomics among these major groups of forest trees.

Integration of SNP genotyping confidence scores in IBD inference

High-throughput single nucleotide polymorphism (SNP) arrays have become the standard platform for linkage and association analyses. The high SNP density of these platforms allows high-resolution identification of ancestral recombination events even for distant relatives many generations apart. However, such inference is sensitive to marker mistyping and current error detection methods rely on the genotyping of additional close relatives. Genotyping algorithms provide a confidence score for each marker call that is currently not integrated in existing methods. There is a need for a model that incorporates this prior information within the standard identical by descent (IBD) and association analyses. We propose a novel model that incorporates marker confidence scores within IBD methods based on the Lander-Green Hidden Markov Model. The novel parameter of this model is the joint distribution of confidence scores and error status per array. We estimate this probability distribution by applying a modified expectation-maximization (EM) procedure on data from nuclear families genotyped with Affymetrix 250K SNP arrays. The converged tables from two different genotyping algorithms are shown for a wide range of error rates. We demonstrate the efficacy of our method in refining the detection of IBD signals using nuclear pedigrees and distant relatives. Plinke, a new version of Plink with an extended pairwise IBD inference model allowing per marker error probabilities is freely available at: http://bioinfo.bgu.ac.il/bsu/software/plinke. obirk@bgu.ac.il; markusb@bgu.ac.il Supplementary data are available at Bioinformatics online.

Immunogenetics of hematopoietic stem cell transplantation: the contribution of microsatellite polymorphism studies

Polymorphisms of short tandem repeats of <10 nucleotides, or microsatellites (Msat), are largely used for post-transplant chimerism analyses in clinical hematopoietic stem cell transplantation (HSCT). Compared to single nucleotide polymorphisms (SNP), they have the advantage of a higher degree of allelic polymorphism and thus a potentially larger degree of informativity. Msat markers contribute to approximately 3% of the human genome and have been highly informative in disease association studies, population genetics, forensic medicine and organ and HSC transplantation. They allowed to expand our knowledge of the haplotypic structure of the HLA complex, including the noncoding sequences in the MHC, and to reach a better characterization of immunological phenotypes. Among the different immunogenetic studies in HSCT patients reviewed here, four Msat loci linked to cytokine genes have been analysed by a number of laboratories as potential candidates markers for HSCT outcome: IFNG, TNFd, IL-10(-1064) and IL-1RN. The low patient numbers and high diversity of clinical parameters account for some heterogeneity of the results. Among the trends starting to emerge from these studies, specific TNFd Msat alleles seem to be associated with acute graft-versus-host disease and mortality. Patient/donor Msat incompatibilities have also been used as surrogate markers to map biologically relevant polymorphisms, with a main focus on MHC-resident genetic variation. High throughput SNP typing and next-generation sequencing technologies will allow acquisition of large-scale genomic data and should allow refined analyses of clinically relevant genotypes in the transplantation settting, although the heterogeneity of the study cohorts will remain an issue. The analysis of Msat polymorphisms may still have a place in functional studies on the impact of Msat diversity in the control of immune response gene expression.

High-throughput single-nucleotide polymorphism discovery and the search for candidate genes for long-term SIVmac nonprogression in Chinese rhesus macaques (Macaca mulatta).

Genetic differences between Indian and Chinese rhesus macaques contribute to the phenotypic variance of clinical trials, including infection with SIVmac. The completion of the rhesus genome has facilitated the discovery of several thousand markers. We developed a genome-wide SNP map for rhesus macaques containing 3869 validated markers with an average distance of 0.88 Mb and used the program VarLD to identify genomic areas with significant differences in linkage disequilibrium (LD) between Indian-derived and Chinese rhesus macaques. Forty-one statistically significant differences in LD between Chinese and Indian-origin rhesus were detected on chromosomes 1, 4, 5 and 11. The region of greatest LD difference was located on the proximal end of chromosome one, which also contained the genes ELAVL4, MAST2 and HIVEP3. These genomic areas provide entry to more detailed studies of gene function. This method is also applicable to the study of differences in biomarkers between regional populations of other species.

Measurements of the Binding of a Large Protein Using a Substrate Density-Controlled DNA Chip

The DNA chip that immobilizes DNA oligonucleotides on a solid plate surface is used for many diagnostic applications. For maximizing the detection sensitivity and accuracy, it is important to control the DNA density on a chip surface and establish a convenient method for optimizing the density. Here, the binding of DNA mismatch-binding protein MutS to the DNA substrate on the chip was investigated, which can be applied for high-throughput single-nucleotide polymorphism analysis in a genome. We prepared the DNA chips where the DNA substrate density was changed simply by using a mixed DNA solution. The binding of MutS was significantly influenced by the amount of DNA substrate on the chip as a consequence of steric crowding, and the moderate density that gave the distance between the DNA substrates greater than the size of the protein was appropriate to obtain accurate kinetic parameters. The substrate density-controlled DNA chip prepared using the mixed DNA solution has distinctive advantages for maximizing the detection capability and kinetic analysis of the binding of MutS and probably also other large proteins.

The development and characterization of a 60K SNP chip for chicken.

BackgroundIn livestock species like the chicken, high throughput single nucleotide polymorphism (SNP) genotyping assays are increasingly being used for whole genome association studies and as a tool in breeding (referred to as genomic selection). To be of value in a wide variety of breeds and populations, the success rate of the SNP genotyping assay, the distribution of the SNP across the genome and the minor allele frequencies (MAF) of the SNPs used are extremely important.ResultsWe describe the design of a moderate density (60k) Illumina SNP BeadChip in chicken consisting of SNPs known to be segregating at high to medium minor allele frequencies (MAF) in the two major types of commercial chicken (broilers and layers). This was achieved by the identification of 352,303 SNPs with moderate to high MAF in 2 broilers and 2 layer lines using Illumina sequencing on reduced representation libraries. To further increase the utility of the chip, we also identified SNPs on sequences currently not covered by the chicken genome assembly (Gallus_gallus-2.1). This was achieved by 454 sequencing of the chicken genome at a depth of 12x and the identification of SNPs on 454-derived contigs not covered by the current chicken genome assembly. In total we added 790 SNPs that mapped to 454-derived contigs as well as 421 SNPs with a position on Chr_random of the current assembly. The SNP chip contains 57,636 SNPs of which 54,293 could be genotyped and were shown to be segregating in chicken populations. Our SNP identification procedure appeared to be highly reliable and the overall validation rate of the SNPs on the chip was 94%. We were able to map 328 SNPs derived from the 454 sequence contigs on the chicken genome. The majority of these SNPs map to chromosomes that are already represented in genome build Gallus_gallus-2.1.0. Twenty-eight SNPs were used to construct two new linkage groups most likely representing two micro-chromosomes not covered by the current genome assembly.ConclusionsThe high success rate of the SNPs on the Illumina chicken 60K Beadchip emphasizes the power of Next generation sequence (NGS) technology for the SNP identification and selection step. The identification of SNPs from sequence contigs derived from NGS sequencing resulted in improved coverage of the chicken genome and the construction of two new linkage groups most likely representing two chicken micro-chromosomes.