Single nucleotide polymorphisms (SNPs) that map to gaps in the human SNP map

An international effort is underway to generate a comprehensive haplotype map (HapMap) of the human genome represented by an estimated 300000 to 1 million ‘tag’ single nucleotide polymorphisms (SNPs). Our analysis indicates that the current human SNP map is not sufficiently dense to support the HapMap project. For example, 24.6% of the genome currently lacks SNPs at the minimal density and spacing that would be required to construct even a conservative tag SNP map containing 300 000 SNPs. In an effort to improve the human SNP map, we identified 140 696 additional SNP candidates using a new bioinformatics pipeline. Over 51 000 of these SNPs mapped to the largest gaps in the human SNP map, leading to significant improvements in these regions. Our SNPs will be immediately useful for the HapMap project, and will allow for the inclusion of many additional genomic intervals in the final HapMap. Nevertheless, our results also indicate that additional SNP discovery projects will be required both to define the haplotype architecture of the human genome and to construct comprehensive tag SNP maps that will be useful for genetic linkage studies in humans.     

Large Genomic Region Free of GWAS-Based Common Variants Contains Fertility-Related Genes

DNA variants, such as single nucleotide polymorphisms (SNPs) and copy number variants (CNVs), are unevenly distributed across the human genome. Currently, dbSNP contains more than 6 million human SNPs, and whole-genome genotyping arrays can assay more than 4 million of them simultaneously. In our study, we first questioned whether published genome-wide association studies (GWASs) assays cover all regions well in the genome. Using dbSNP build 135 data, we identified 50 genomic regions longer than 100 Kb that do not contain any common SNPs, i.e., those with minor allele frequency (MAF)≥1%. Secondly, because conserved regions are generally of functional importance, we tested genes in those large genomic regions without common SNPs. We found 97 genes and were enriched for reproduction function. In addition, we further filtered out regions with CNVs listed in the Database of Genomic Variants (DGV), segmental duplications from Human Genome Project and common variants identified by personal genome sequencing (UCSC). No region survived after those filtering. Our analysis suggests that, while there may not be many large genomic regions free of common variants, there are still some “holes” in the current human genomic map for common SNPs. Because GWAS only focused on common SNPs, interpretation of GWAS results should take this limitation into account. Particularly, two recent GWAS of fertility may be incomplete due to the map deficit. Additional SNP discovery efforts should pay close attention to these regions.     

Sequence-based ultra-dense genetic and physical maps reveal structural variations of allopolyploid cotton genomes

BackgroundSNPs are the most abundant polymorphism type, and have been explored in many crop genomic studies, including rice and maize. SNP discovery in allotetraploid cotton genomes has lagged behind that of other crops due to their complexity and polyploidy. In this study, genome-wide SNPs are detected systematically using next-generation sequencing and efficient SNP genotyping methods, and used to construct a linkage map and characterize the structural variations in polyploid cotton genomes.ResultsWe construct an ultra-dense inter-specific genetic map comprising 4,999,048 SNP loci distributed unevenly in 26 allotetraploid cotton linkage groups and covering 4,042 cM. The map is used to order tetraploid cotton genome scaffolds for accurate assembly of G. hirsutum acc. TM-1. Recombination rates and hotspots are identified across the cotton genome by comparing the assembled draft sequence and the genetic map. Using this map, genome rearrangements and centromeric regions are identified in tetraploid cotton by combining information from the publicly-available G. raimondii genome with fluorescent in situ hybridization analysis.ConclusionsWe report the genotype-by-sequencing method used to identify millions of SNPs between G. hirsutum and G. barbadense. We construct and use an ultra-dense SNP map to correct sequence mis-assemblies, merge scaffolds into pseudomolecules corresponding to chromosomes, detect genome rearrangements, and identify centromeric regions in allotetraploid cottons. We find that the centromeric retro-element sequence of tetraploid cotton derived from the D subgenome progenitor might have invaded the A subgenome centromeres after allotetrapolyploid formation. This study serves as a valuable genomic resource for genetic research and breeding of cotton.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0678-1) contains supplementary material, which is available to authorized users.     

Privacy Risks from Genomic Data-Sharing Beacons

The human genetics community needs robust protocols that enable secure sharing of genomic data from participants in genetic research. Beacons are web servers that answer allele-presence queries—such as “Do you have a genome that has a specific nucleotide (e.g., A) at a specific genomic position (e.g., position 11,272 on chromosome 1)?”—with either “yes” or “no.” Here, we show that individuals in a beacon are susceptible to re-identification even if the only data shared include presence or absence information about alleles in a beacon. Specifically, we propose a likelihood-ratio test of whether a given individual is present in a given genetic beacon. Our test is not dependent on allele frequencies and is the most powerful test for a specified false-positive rate. Through simulations, we showed that in a beacon with 1,000 individuals, re-identification is possible with just 5,000 queries. Relatives can also be identified in the beacon. Re-identification is possible even in the presence of sequencing errors and variant-calling differences. In a beacon constructed with 65 European individuals from the 1000 Genomes Project, we demonstrated that it is possible to detect membership in the beacon with just 250 SNPs. With just 1,000 SNP queries, we were able to detect the presence of an individual genome from the Personal Genome Project in an existing beacon. Our results show that beacons can disclose membership and implied phenotypic information about participants and do not protect privacy a priori. We discuss risk mitigation through policies and standards such as not allowing anonymous pings of genetic beacons and requiring minimum beacon sizes.     

Genome Wide Association Study Identifies New Loci Associated with Undesired Coat Color Phenotypes in Saanen Goats

This paper reports a quantitative genetics and genomic analysis of undesirable coat color patterns in goats. Two undesirable coat colors have routinely been recorded for the past 15 years in French Saanen goats. One fifth of Saanen females have been phenotyped “pink” (8.0%) or “pink neck” (11.5%) and consequently have not been included in the breeding program as elite animals. Heritability of the binary “pink” and “pink neck” phenotype, estimated from 103,443 females was 0.26 for “pink” and 0.21 for “pink neck”. Genome wide association studies (using haplotypes or single SNPs) were implemented using a daughter design of 810 Saanen goats sired by 9 Artificial Insemination bucks genotyped with the goatSNP50 chip. A highly significant signal (-log10pvalue = 10.2) was associated with the “pink neck” phenotype on chromosome 11, suggesting the presence of a major gene. Highly significant signals for the “pink” phenotype were found on chromosomes 5 and 13 (-log10p values of 7.2 and, 7.7 respectively). The most significant SNP on chromosome 13 was in the ASIP gene region, well known for its association with coat color phenotypes. Nine significant signals were also found for both traits. The highest signal for each trait was detected by both single SNP and haplotype approaches, whereas the smaller signals were not consistently detected by the two methods. Altogether these results demonstrated a strong genetic control of the “pink” and “pink neck” phenotypes in French Saanen goats suggesting that SNP information could be used to identify and remove undesired colored animals from the breeding program.     

Comparison of a high-density genetic linkage map to genome features in the model grass <Emphasis Type="Italic">Brachypodium distachyon</Emphasis>

The small annual grass Brachypodium distachyon (Brachypodium) is rapidly emerging as a powerful model system to study questions unique to the grasses. Many Brachypodium resources have been developed including a whole genome sequence, highly efficient transformation and a large germplasm collection. We developed a genetic linkage map of Brachypodium using single nucleotide polymorphism (SNP) markers and an F₂ mapping population of 476 individuals. SNPs were identified by targeted resequencing of single copy genomic sequences. Using the Illumina GoldenGate Genotyping platform we placed 558 markers into five linkage groups corresponding to the five chromosomes of Brachypodium. The unusually long total genetic map length, 1,598 centiMorgans (cM), indicates that the Brachypodium mapping population has a high recombination rate. By comparing the genetic map to genome features we found that the recombination rate was positively correlated with gene density and negatively correlated with repetitive regions and sites of ancestral chromosome fusions that retained centromeric repeat sequences. A comparison of adjacent genome regions with high versus low recombination rates revealed a positive correlation between interspecific synteny and recombination rate.     

The effect of SNP discovery method and sample size on estimation of population genetic data for Chinese and Indian rhesus macaques (<Emphasis Type="Italic">Macaca mulatta</Emphasis>)

This study was designed to address issues regarding sample size and marker location that have arisen from the discovery of SNPs in the genomes of poorly characterized primate species and the application of these markers to the study of primate population genetics. We predict the effect of discovery sample size on the probability of discovering both rare and common SNPs and then compare this prediction with the proportion of common and rare SNPs discovered when different numbers of individuals are sequenced. Second, we examine the effect of genomic region on estimates of common population genetic data, comparing markers from both coding and non-coding regions of the rhesus macaque genome and the population genetic data calculated from these markers, to measure the degree and direction of bias introduced by SNPs located in coding versus non-coding regions of the genome. We found that both discovery sample size and genomic region surveyed affect SNP marker attributes and population genetic estimates, even when these are calculated from an expanded data set containing more individuals than the original discovery data set. Although none of the SNP detection methods or genomic regions tested in this study was completely uninformative, these results show that each has a different kind of genetic variation that is suitable for different purposes, and each introduces specific types of bias. Given that each SNP marker has an individual evolutionary history, we calculated that the most complete and unbiased representation of the genetic diversity present in the individual can be obtained by incorporating at least 10 individuals into the discovery sample set, to ensure the discovery of both common and rare polymorphisms.     

SNP Discovery and Chromosome Anchoring Provide the First Physically-Anchored Hexaploid Oat Map and Reveal Synteny with Model Species

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.     

Comprehensive Characterization of Human Genome Variation by High Coverage Whole-Genome Sequencing of Forty Four Caucasians

Whole genome sequencing studies are essential to obtain a comprehensive understanding of the vast pattern of human genomic variations. Here we report the results of a high-coverage whole genome sequencing study for 44 unrelated healthy Caucasian adults, each sequenced to over 50-fold coverage (averaging 65.8×). We identified approximately 11 million single nucleotide polymorphisms (SNPs), 2.8 million short insertions and deletions, and over 500,000 block substitutions. We showed that, although previous studies, including the 1000 Genomes Project Phase 1 study, have catalogued the vast majority of common SNPs, many of the low-frequency and rare variants remain undiscovered. For instance, approximately 1.4 million SNPs and 1.3 million short indels that we found were novel to both the dbSNP and the 1000 Genomes Project Phase 1 data sets, and the majority of which (∼96%) have a minor allele frequency less than 5%. On average, each individual genome carried ∼3.3 million SNPs and ∼492,000 indels/block substitutions, including approximately 179 variants that were predicted to cause loss of function of the gene products. Moreover, each individual genome carried an average of 44 such loss-of-function variants in a homozygous state, which would completely “knock out” the corresponding genes. Across all the 44 genomes, a total of 182 genes were “knocked-out” in at least one individual genome, among which 46 genes were “knocked out” in over 30% of our samples, suggesting that a number of genes are commonly “knocked-out” in general populations. Gene ontology analysis suggested that these commonly “knocked-out” genes are enriched in biological process related to antigen processing and immune response. Our results contribute towards a comprehensive characterization of human genomic variation, especially for less-common and rare variants, and provide an invaluable resource for future genetic studies of human variation and diseases.     

Rapid SNP Discovery and a RAD-Based High-Density Linkage Map in Jujube (Ziziphus Mill.)

Background

Ziziphus Mill. (jujube), the most valued genus of Rhamnaceae, comprises of a number of economically and ecologically important species such as Z. jujuba Mill., Z. acidojujuba Cheng et Liu and Z. mauritiana Lam. Single nucleotide polymorphism (SNP) markers and a high-density genetic map are of great benefit to the improvement of the crop, mapping quantitative trait loci (QTL) and analyzing genome structure. However, such a high-density map is still absent in the genus Ziziphus and even the family Rhamnaceae. The recently developed restriction-site associated DNA (RAD) marker has been proven to be most powerful in genetic map construction. The objective of this study was to construct a high-density linkage map using the RAD tags generated by next generation sequencing.

Results

An interspecific F1 population and their parents (Z. jujuba Mill. ‘JMS2’ × Z. acidojujuba Cheng et Liu ‘Xing 16’) were genotyped using a mapping-by-sequencing approach, to generate RAD-based SNP markers. A total of 42,784 putative high quality SNPs were identified between the parents and 2,872 high-quality RAD markers were grouped in genetic maps. Of the 2,872 RAD markers, 1,307 were linked to the female genetic map, 1,336 to the male map, and 2,748 to the integrated map spanning 913.87 centi-morgans (cM) with an average marker interval of 0.34 cM. The integrated map contained 12 linkage groups (LGs), consistent with the haploid chromosome number of the two parents.

Conclusion

We first generated a high-density genetic linkage map with 2,748 RAD markers for jujube and a large number of SNPs were also developed. It provides a useful tool for both marker-assisted breeding and a variety of genome investigations in jujube, such as sequence assembly, gene localization, QTL detection and genome structure comparison.     

<Emphasis Type="Italic">In silico</Emphasis> physical mapping software for the <Emphasis Type="Italic">Triticum aestivum</Emphasis> genome

The large size of the Triticum aestivum genome makes it unlikely that a complete genome sequence for wheat will be available in the near future. Exploiting the conserved genome organization between wheat and rice and existing genomic resources, we have constructed in silico physical mapping software for wheat, assigning a gross physical location(s) into chromosome bins to 22,626 representative wheat gene sequences. To validate the predictions from the software we compared the predicted locations of ten ESTs to their positions experimentally determined by SNP marker analysis. Six of the sequences were correctly positioned on the map including four that demonstrated a high level of colinearity with their orthologous rice genomic region. This tool will facilitate the development of molecular markers for regions of interest and the creation of map-based cloning strategies in areas demonstrating high levels of sequence conservation and organization between wheat and rice.     

SNP identification in unamplified human genomic DNA with gold nanoparticle probes

Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome. SNPs may be linked to genetic predispositions, frank disorders or adverse drug responses, or they may serve as genetic markers in linkage disequilibrium analysis. Thus far, established SNP detection techniques have utilized enzymes to meet the sensitivity and specificity requirements needed to overcome the high complexity of the human genome. Herein, we present for the first time a microarray-based method that allows multiplex SNP genotyping in total human genomic DNA without the need for target amplification or complexity reduction. This direct SNP genotyping methodology requires no enzymes and relies on the high sensitivity of the gold nanoparticle probes. Specificity is derived from two sequential oligonucleotide hybridizations to the target by allele-specific surface-immobilized capture probes and gene-specific oligonucleotide-functionalized gold nanoparticle probes. Reproducible multiplex SNP detection is demonstrated with unamplified human genomic DNA samples representing all possible genotypes for three genes involved in thrombotic disorders. The assay format is simple, rapid and robust pointing to its suitability for multiplex SNP profiling at the ‘point of care’.     

An EST-derived SNP and SSR genetic linkage map of cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz) is one of the most important food security crops in the tropics and increasingly being adopted for agro-industrial processing. Genetic improvement of cassava can be enhanced through marker-assisted breeding. For this, appropriate genomic tools are required to dissect the genetic architecture of economically important traits. Here, a genome-wide SNP-based genetic map of cassava anchored in SSRs is presented. An outbreeder full-sib (F1) family was genotyped on two independent SNP assay platforms: an array of 1,536 SNPs on Illumina's GoldenGate platform was used to genotype a first batch of 60 F1. Of the 1,358 successfully converted SNPs, 600 which were polymorphic in at least one of the parents and was subsequently converted to KBiosciences' KASPar assay platform for genotyping 70 additional F1. High-precision genotyping of 163 informative SSRs using capillary electrophoresis was also carried out. Linkage analysis resulted in a final linkage map of 1,837 centi-Morgans (cM) containing 568 markers (434 SNPs and 134 SSRs) distributed across 19 linkage groups. The average distance between adjacent markers was 3.4?cM. About 94.2% of the mapped SNPs and SSRs have also been localized on scaffolds of version 4.1 assembly of the cassava draft genome sequence. This more saturated genetic linkage map of cassava that combines SSR and SNP markers should find several applications in the improvement of cassava including aligning scaffolds of the cassava genome sequence, genetic analyses of important agro-morphological traits, studying the linkage disequilibrium landscape and comparative genomics.     

Transcriptome-based single nucleotide polymorphism markers for genome mapping in Japanese pear (Pyrus pyrifolia Nakai)

The development of single nucleotide polymorphism (SNP) markers in Japanese pear (Pyrus pyrifolia Nakai) offers the opportunity to use DNA markers for marker-assisted selection in breeding programs because of their high abundance, codominant inheritance, and potential for automated high-throughput analysis. We developed a 1,536-SNP bead array without a reference genome sequence from more than 44,000 base changes on the basis of a large-scale expressed sequence tag (EST) analysis combined with 454 genome sequencing data of Japanese pear ‘Housui’. Among the 1,536 SNPs on the array, 756 SNPs were genotyped, and 609 SNP loci were mapped to linkage groups on a genetic linkage map of ‘Housui’, based on progeny of an interspecific cross between European pear (Pyrus communis L.) ‘Bartlett’ and ‘Housui’. The newly constructed genetic linkage map consists of 951 loci, comprising 609 new SNPs, 110 pear genomic simple sequence repeats (SSRs), 25 pear EST–SSRs, 127 apple SSRs, 61 pear SNPs identified by the “potential intron polymorphism” method, and 19 other loci. The map covers 22 linkage groups spanning 1341.9 cM with an average distance of 1.41 cM between markers and is anchored to reference genetic linkage maps of European pears and apples. A total of 514 contigs containing mapped SNP loci showed significant similarity to known proteins by functional annotation analysis.     

A ddRAD Based Linkage Map of the Cultivated Strawberry,Fragaria xananassa

The cultivated strawberry (Fragaria ×ananassa Duch.) is an allo-octoploid considered difficult to disentangle genetically due to its four relatively similar sub-genomic chromosome sets. This has been alleviated by the recent release of the strawberry IStraw90 whole genome genotyping array. However, array resolution relies on the genotypes used in the array construction and may be of limited general use. SNP detection based on reduced genomic sequencing approaches has the potential of providing better coverage in cases where the studied genotypes are only distantly related from the SNP array’s construction foundation. Here we have used double digest restriction-associated DNA sequencing (ddRAD) to identify SNPs in a 145 seedling F₁ hybrid population raised from the cross between the cultivars Sonata (♀) and Babette (♂). A linkage map containing 907 markers which spanned 1,581.5 cM across 31 linkage groups representing the 28 chromosomes of the species. Comparing the physical span of the SNP markers with the F. vesca genome sequence, the linkage groups resolved covered 79% of the estimated 830 Mb of the F. ×ananassa genome. Here, we have developed the first linkage map for F. ×ananassa using ddRAD and show that this technique and other related techniques are useful tools for linkage map development and downstream genetic studies in the octoploid strawberry.     

Use of bovine EST data and human genomic sequences to map 100 gene-specific bovine markers

A system to use bovine EST data in conjunction with human genomic sequence to improve the bovine linkage map over the entire genome or on specific chromosomes was evaluated. Bovine EST sequence was used to provide primer sequences corresponding to bovine genes, while human genomic sequence directed primer design to flank introns and produce amplicons of appropriate size for efficient direct sequencing. The sequence tagged sites (STS) produced in this way from the four sires of the MARC reference families were examined for single nucleotide polymorphisms (SNPs) that could be used to map the corresponding genes. With this approach, along with a primer/extension mass spectrometry SNP genotyping assay, 100 ESTs were placed on the bovine genetic linkage map. The first 70 were chosen at random from bovine EST–human genomic comparisons. An additional 30 ESTs were successfully mapped to bovine Chromosome 19 (BTA19), and comparison of the resulting BTA19 map to the position of the corresponding human orthologs on the HSA17 draft sequences revealed differences in the spacing and order of genes. Over 80% of successful amplicons contained SNPs, indicating that this is an efficient approach to generating EST-associated genetic markers. We have demonstrated the feasibility of constructing a linkage map based on SNPs associated with ESTs and the plausibility of utilizing EST, comparative mapping information, and human sequence data to target regions of the bovine genome for SNP marker development.     

BAC-HAPPY Mapping (BAP Mapping): A New and Efficient Protocol for Physical Mapping

Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical “contig” maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning ∼10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual “BAC-HAPPY-mapping” to convert BAC landing data into BAC linkage contigs is possible.