Identification of Genome-Wide Copy Number Variations among Diverse Pig Breeds Using SNP Genotyping Arrays

Copy number variations (CNVs) are important forms of genetic variation complementary to SNPs, and can be considered as promising markers for some phenotypic and economically important traits or diseases susceptibility in domestic animals. In the present study, we performed a genome-wide CNV identification in 14 individuals selected from diverse populations, including six types of Chinese indigenous breeds, one Asian wild boar population, as well as three modern commercial foreign breeds. We identified 63 CNVRs in total, which covered 9.98 Mb of polymorphic sequence and corresponded to 0.36% of the genome sequence. The length of these CNVRs ranged from 3.20 to 827.21 kb, with an average of 158.37 kb and a median of 97.85 kb. Functional annotation revealed these identified CNVR have important molecular function, and may play an important role in exploring the genetic basis of phenotypic variability and disease susceptibility among pigs. Additionally, to confirm these potential CNVRs, we performed qPCR for 12 randomly selected CNVRs and 8 of them (66.67%) were confirmed successfully. CNVs detected in diverse populations herein are essential complementary to the CNV map in the pig genome, which provide an important resource for studies of genomic variation and the association between various economically important traits and CNVs.     

Analysis of Genome-Wide Copy Number Variations in Chinese Indigenous and Western Pig Breeds by 60 K SNP Genotyping Arrays

Copy number variations (CNVs) represent a substantial source of structural variants in mammals and contribute to both normal phenotypic variability and disease susceptibility. Although low-resolution CNV maps are produced in many domestic animals, and several reports have been published about the CNVs of porcine genome, the differences between Chinese and western pigs still remain to be elucidated. In this study, we used Porcine SNP60 BeadChip and PennCNV algorithm to perform a genome-wide CNV detection in 302 individuals from six Chinese indigenous breeds (Tongcheng, Laiwu, Luchuan, Bama, Wuzhishan and Ningxiang pigs), three western breeds (Yorkshire, Landrace and Duroc) and one hybrid (Tongcheng×Duroc). A total of 348 CNV Regions (CNVRs) across genome were identified, covering 150.49 Mb of the pig genome or 6.14% of the autosomal genome sequence. In these CNVRs, 213 CNVRs were found to exist only in the six Chinese indigenous breeds, and 60 CNVRs only in the three western breeds. The characters of CNVs in four Chinese normal size breeds (Luchuan, Tongcheng and Laiwu pigs) and two minipig breeds (Bama and Wuzhishan pigs) were also analyzed in this study. Functional annotation suggested that these CNVRs possess a great variety of molecular function and may play important roles in phenotypic and production traits between Chinese and western breeds. Our results are important complementary to the CNV map in pig genome, which provide new information about the diversity of Chinese and western pig breeds, and facilitate further research on porcine genome CNVs.     

Genome-Wide Mapping of Structural Variations Reveals a Copy Number Variant That Determines Reproductive Morphology in Cucumber

Structural variations (SVs) represent a major source of genetic diversity. However, the functional impact and formation mechanisms of SVs in plant genomes remain largely unexplored. Here, we report a nucleotide-resolution SV map of cucumber (Cucumis sativas) that comprises 26,788 SVs based on deep resequencing of 115 diverse accessions. The largest proportion of cucumber SVs was formed through nonhomologous end-joining rearrangements, and the occurrence of SVs is closely associated with regions of high nucleotide diversity. These SVs affect the coding regions of 1676 genes, some of which are associated with cucumber domestication. Based on the map, we discovered a copy number variation (CNV) involving four genes that defines the Female (F) locus and gives rise to gynoecious cucumber plants, which bear only female flowers and set fruit at almost every node. The CNV arose from a recent 30.2-kb duplication at a meiotically unstable region, likely via microhomology-mediated break-induced replication. The SV set provides a snapshot of structural variations in plants and will serve as an important resource for exploring genes underlying key traits and for facilitating practical breeding in cucumber.     

Unravelling the Complexity of Human Olfactory Receptor Repertoire by Copy Number Analysis across Population Using High Resolution Arrays

Olfactory receptors (OR), responsible for detection of odor molecules, belong to the largest family of genes and are highly polymorphic in nature having distinct polymorphisms associated with specific regions around the globe. Since there are no reports on the presence of copy number variations in OR repertoire of Indian population, the present investigation in 43 Indians along with 270 HapMap and 31 Tibetan samples was undertaken to study genome variability and evolution. Analysis was performed using Affymetrix Genome-Wide Human SNP Array 6.0 chip, Affymterix CytoScan^® High-Density array, HD-CNV, and MAFFT program. We observed a total of 1527 OR genes in 503 CNV events from 81.3% of the study group, which includes 67.6% duplications and 32.4% deletions encompassing more of genes than pseudogenes. We report human genotypic variation in functional OR repertoire size across populations and it was found that the combinatorial effect of both “orthologous obtained from closely related species” and “paralogous derived sequences” provide the complexity to the continuously occurring OR CNVs.     

Identification of Networks of Co-Occurring,Tumor-Related DNA Copy Number Changes Using a Genome-Wide Scoring Approach

Tumorigenesis is a multi-step process in which normal cells transform into malignant tumors following the accumulation of genetic mutations that enable them to evade the growth control checkpoints that would normally suppress their growth or result in apoptosis. It is therefore important to identify those combinations of mutations that collaborate in cancer development and progression. DNA copy number alterations (CNAs) are one of the ways in which cancer genes are deregulated in tumor cells. We hypothesized that synergistic interactions between cancer genes might be identified by looking for regions of co-occurring gain and/or loss. To this end we developed a scoring framework to separate truly co-occurring aberrations from passenger mutations and dominant single signals present in the data. The resulting regions of high co-occurrence can be investigated for between-region functional interactions. Analysis of high-resolution DNA copy number data from a panel of 95 hematological tumor cell lines correctly identified co-occurring recombinations at the T-cell receptor and immunoglobulin loci in T- and B-cell malignancies, respectively, showing that we can recover truly co-occurring genomic alterations. In addition, our analysis revealed networks of co-occurring genomic losses and gains that are enriched for cancer genes. These networks are also highly enriched for functional relationships between genes. We further examine sub-networks of these networks, core networks, which contain many known cancer genes. The core network for co-occurring DNA losses we find seems to be independent of the canonical cancer genes within the network. Our findings suggest that large-scale, low-intensity copy number alterations may be an important feature of cancer development or maintenance by affecting gene dosage of a large interconnected network of functionally related genes.     

A Bayesian Analysis for Identifying DNA Copy Number Variations Using a Compound Poisson Process

To study chromosomal aberrations that may lead to cancer formation or genetic diseases, the array-based Comparative Genomic Hybridization (aCGH) technique is often used for detecting DNA copy number variants (CNVs). Various methods have been developed for gaining CNVs information based on aCGH data. However, most of these methods make use of the log-intensity ratios in aCGH data without taking advantage of other information such as the DNA probe (e.g., biomarker) positions/distances contained in the data. Motivated by the specific features of aCGH data, we developed a novel method that takes into account the estimation of a change point or locus of the CNV in aCGH data with its associated biomarker position on the chromosome using a compound Poisson process. We used a Bayesian approach to derive the posterior probability for the estimation of the CNV locus. To detect loci of multiple CNVs in the data, a sliding window process combined with our derived Bayesian posterior probability was proposed. To evaluate the performance of the method in the estimation of the CNV locus, we first performed simulation studies. Finally, we applied our approach to real data from aCGH experiments, demonstrating its applicability.     

Population Analysis of Large Copy Number Variants and Hotspots of Human Genetic Disease

Copy number variants (CNVs) contribute to human genetic and phenotypic diversity. However, the distribution of larger CNVs in the general population remains largely unexplored. We identify large variants in ~2500 individuals by using Illumina SNP data, with an emphasis on “hotspots” prone to recurrent mutations. We find variants larger than 500 kb in 5%–10% of individuals and variants greater than 1 Mb in 1%–2%. In contrast to previous studies, we find limited evidence for stratification of CNVs in geographically distinct human populations. Importantly, our sample size permits a robust distinction between truly rare and polymorphic but low-frequency copy number variation. We find that a significant fraction of individual CNVs larger than 100 kb are rare and that both gene density and size are strongly anticorrelated with allele frequency. Thus, although large CNVs commonly exist in normal individuals, which suggests that size alone can not be used as a predictor of pathogenicity, such variation is generally deleterious. Considering these observations, we combine our data with published CNVs from more than 12,000 individuals contrasting control and neurological disease collections. This analysis identifies known disease loci and highlights additional CNVs (e.g., 3q29, 16p12, and 15q25.2) for further investigation. This study provides one of the first analyses of large, rare (0.1%–1%) CNVs in the general population, with insights relevant to future analyses of genetic disease.     

A Genome-Wide Association Study in Large White and Landrace Pig Populations for Number Piglets Born Alive

The number of piglets born alive (NBA) per litter is one of the most important traits in pig breeding due to its influence on production efficiency. It is difficult to improve NBA because the heritability of the trait is low and it is governed by a high number of loci with low to moderate effects. To clarify the biological and genetic background of NBA, genome-wide association studies (GWAS) were performed using 4,012 Large White and Landrace pigs from herdbook and commercial breeding companies in Germany (3), Austria (1) and Switzerland (1). The animals were genotyped with the Illumina PorcineSNP60 BeadChip. Because of population stratifications within and between breeds, clusters were formed using the genetic distances between the populations. Five clusters for each breed were formed and analysed by GWAS approaches. In total, 17 different significant markers affecting NBA were found in regions with known effects on female reproduction. No overlapping significant chromosome areas or QTL between Large White and Landrace breed were detected.     

Identification of Rare Recurrent Copy Number Variants in High-Risk Autism Families and Their Prevalence in a Large ASD Population

Structural variation is thought to play a major etiological role in the development of autism spectrum disorders (ASDs), and numerous studies documenting the relevance of copy number variants (CNVs) in ASD have been published since 2006. To determine if large ASD families harbor high-impact CNVs that may have broader impact in the general ASD population, we used the Affymetrix genome-wide human SNP array 6.0 to identify 153 putative autism-specific CNVs present in 55 individuals with ASD from 9 multiplex ASD pedigrees. To evaluate the actual prevalence of these CNVs as well as 185 CNVs reportedly associated with ASD from published studies many of which are insufficiently powered, we designed a custom Illumina array and used it to interrogate these CNVs in 3,000 ASD cases and 6,000 controls. Additional single nucleotide variants (SNVs) on the array identified 25 CNVs that we did not detect in our family studies at the standard SNP array resolution. After molecular validation, our results demonstrated that 15 CNVs identified in high-risk ASD families also were found in two or more ASD cases with odds ratios greater than 2.0, strengthening their support as ASD risk variants. In addition, of the 25 CNVs identified using SNV probes on our custom array, 9 also had odds ratios greater than 2.0, suggesting that these CNVs also are ASD risk variants. Eighteen of the validated CNVs have not been reported previously in individuals with ASD and three have only been observed once. Finally, we confirmed the association of 31 of 185 published ASD-associated CNVs in our dataset with odds ratios greater than 2.0, suggesting they may be of clinical relevance in the evaluation of children with ASDs. Taken together, these data provide strong support for the existence and application of high-impact CNVs in the clinical genetic evaluation of children with ASD.     

Digital Genotyping of Macrosatellites and Multicopy Genes Reveals Novel Biological Functions Associated with Copy Number Variation of Large Tandem Repeats

Tandem repeats are common in eukaryotic genomes, but due to difficulties in assaying them remain poorly studied. Here, we demonstrate the utility of Nanostring technology as a targeted approach to perform accurate measurement of tandem repeats even at extremely high copy number, and apply this technology to genotype 165 HapMap samples from three different populations and five species of non-human primates. We observed extreme variability in copy number of tandemly repeated genes, with many loci showing 5–10 fold variation in copy number among humans. Many of these loci show hallmarks of genome assembly errors, and the true copy number of many large tandem repeats is significantly under-represented even in the high quality ‘finished’ human reference assembly. Importantly, we demonstrate that most large tandem repeat variations are not tagged by nearby SNPs, and are therefore essentially invisible to SNP-based GWAS approaches. Using association analysis we identify many cis correlations of large tandem repeat variants with nearby gene expression and DNA methylation levels, indicating that variations of tandem repeat length are associated with functional effects on the local genomic environment. This includes an example where expansion of a macrosatellite repeat is associated with increased DNA methylation and suppression of nearby gene expression, suggesting a mechanism termed “repeat induced gene silencing”, which has previously been observed only in transgenic organisms. We also observed multiple signatures consistent with altered selective pressures at tandemly repeated loci, suggesting important biological functions. Our studies show that tandemly repeated loci represent a highly variable fraction of the genome that have been systematically ignored by most previous studies, copy number variation of which can exert functionally significant effects. We suggest that future studies of tandem repeat loci will lead to many novel insights into their role in modulating both genomic and phenotypic diversity.     

Genome-Wide Copy Number Variation in Sporadic Amyotrophic Lateral Sclerosis in the Turkish Population: Deletion of EPHA3 Is a Possible Protective Factor

The genome-wide presence of copy number variations (CNVs), which was shown to affect the expression and function of genes, has been recently suggested to confer risk for various human disorders, including Amyotrophic Lateral Sclerosis (ALS). We have performed a genome-wide CNV analysis using PennCNV tool and 733K GWAS data of 117 Turkish ALS patients and 109 matched healthy controls. Case-control association analyses have implicated the presence of both common (>5%) and rare (<5%) CNVs in the Turkish population. In the framework of this study, we identified several common and rare loci that may have an impact on ALS pathogenesis. None of the CNVs associated has been implicated in ALS before, but some have been reported in different types of cancers and autism. The most significant associations were shown for 41 kb and 15 kb intergenic heterozygous deletions (Chr11: 50,545,009–50,586,426 and Chr19: 20,860,930–20,875,787) both contributing to increased risk for ALS. CNVs in coding regions of the MAP4K3, HLA-B, EPHA3 and DPYD genes were detected however, after validation by Log R Ratio (LRR) values and TaqMan CNV genotyping, only EPHA3 deletion remained as a potential protective factor for ALS (p = 0.0065024). Based on the knowledge that EPHA4 has been previously shown to rescue SOD1 transgenic mice from ALS phenotype and prolongs survival, EPHA3 may be a promising candidate for therepuetic interventions.     

Outlier-Based Identification of Copy Number Variations Using Targeted Resequencing in a Small Cohort of Patients with Tetralogy of Fallot

Copy number variations (CNVs) are one of the main sources of variability in the human genome. Many CNVs are associated with various diseases including cardiovascular disease. In addition to hybridization-based methods, next-generation sequencing (NGS) technologies are increasingly used for CNV discovery. However, respective computational methods applicable to NGS data are still limited. We developed a novel CNV calling method based on outlier detection applicable to small cohorts, which is of particular interest for the discovery of individual CNVs within families, de novo CNVs in trios and/or small cohorts of specific phenotypes like rare diseases. Approximately 7,000 rare diseases are currently known, which collectively affect ∼6% of the population. For our method, we applied the Dixon’s Q test to detect outliers and used a Hidden Markov Model for their assessment. The method can be used for data obtained by exome and targeted resequencing. We evaluated our outlier- based method in comparison to the CNV calling tool CoNIFER using eight HapMap exome samples and subsequently applied both methods to targeted resequencing data of patients with Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease. In both the HapMap samples and the TOF cases, our method is superior to CoNIFER, such that it identifies more true positive CNVs. Called CNVs in TOF cases were validated by qPCR and HapMap CNVs were confirmed with available array-CGH data. In the TOF patients, we found four copy number gains affecting three genes, of which two are important regulators of heart development (NOTCH1, ISL1) and one is located in a region associated with cardiac malformations (PRODH at 22q11). In summary, we present a novel CNV calling method based on outlier detection, which will be of particular interest for the analysis of de novo or individual CNVs in trios or cohorts up to 30 individuals, respectively.     

Quantification of Normal Cell Fraction and Copy Number Neutral LOH in Clinical Lung Cancer Samples Using SNP Array Data

Background

Technologies based on DNA microarrays have the potential to provide detailed information on genomic aberrations in tumor cells. In practice a major obstacle for quantitative detection of aberrations is the heterogeneity of clinical tumor tissue. Since tumor tissue invariably contains genetically normal stromal cells, this may lead to a failure to detect aberrations in the tumor cells.

Principal Finding

Using SNP array data from 44 non-small cell lung cancer samples we have developed a bioinformatic algorithm that accurately models the fractions of normal and tumor cells in clinical tumor samples. The proportion of normal cells in combination with SNP array data can be used to detect and quantify copy number neutral loss-of-heterozygosity (CNNLOH) in the tumor cells both in crude tumor tissue and in samples enriched for tumor cells by laser capture microdissection.

Conclusion

Genome-wide quantitative analysis of CNNLOH using the CNNLOH Quantifier method can help to identify recurrent aberrations contributing to tumor development in clinical tumor samples. In addition, SNP-array based analysis of CNNLOH may become important for detection of aberrations that can be used for diagnostic and prognostic purposes.     

三色荧光技术在SNP检测中的应用

利用三色荧光标记的A、C、T双脱氧核苷酸单碱基延伸的方法结合编码寡核苷酸芯片技术检测单核苷酸多态性 (SNP)的基因型。以beta地中海贫血样本基因 (HBB基因 )突变作为模型的研究结果显示该方法能同时对多位点的SNP进行检测。     

Prokaryotic Genome Size and SSU rDNA Copy Number: Estimation of Microbial Relative Abundance from a Mixed Population

Abstract Determination of the relative abundance of a specific prokaryote in an environmental sample is of major interest in applied and environmental microbiology. Relative abundance can be calculated using knowledge of SSU rDNA copy number, amount of SSU rDNA in the sample, and a weighted average estimate of the genome sizes for organisms in the original sample. By surveying the literature, we provide estimates of genome size and SSU rDNA copy number for 303 and 101 prokaryotes, respectively. This compilation can be used to make reasonable estimates for a wide range of organisms in the calculation of relative abundance. A statistical analysis suggests that no correlation exists between genome size and SSU rDNA copy number. A phylogenetic analysis is used to offer insights into the evolution of both genome size and SSU rDNA copy number. Received: 29 January 1999; Accepted: 29 April 1999     

基于数字PCR的不同品种鸭组织中线粒体与核DNA拷贝数差异研究

肉和肉制品是人类生活的重要营养来源,但近年来肉制品中发生的掺假使假事件屡见不鲜,使得肉品的质量安全问题已经成为全世界关注的热点话题。以核酸为目标的动物源鉴定是当前普遍使用的方法。在核酸检测中,常用线粒体基因或核基因作为靶标,缺乏统一标准。以绍兴鸭和北京鸭等不同品种及生鲜组织(鸭血、鸭胸肉、鸭肝、鸭皮、鸭心和鸭腿肉)为实验材料,提取DNA后利用微滴式数字PCR开展线粒体和核DNA拷贝数的比较研究,以两者拷贝数及其比值的变异系数为判定依据。结果显示,核DNA的拷贝数在不同品种鸭组织间相对稳定,且变异系数小于线粒体DNA,表明核DNA是开展鸭肉制品掺假定量检测的最适DNA来源。鸭腿肉中线粒体/核DNA拷贝数比值的变异系数最小,表明线粒体DNA作为靶基因的鸭肉掺假比例定量检测时,鸭腿肉来源的肉制品是最佳选择。     

Mapping a Large Number of QTL for Durable Resistance to Stripe Rust in Winter Wheat Druchamp Using SSR and SNP Markers

Winter wheat Druchamp has both high-temperature adult-plant (HTAP) resistance and all-stage resistance to stripe rust caused by Puccinia striiformis f. sp. tritici (Pst). The HTAP resistance in Druchamp is durable as the variety has been resistant in adult-plant stage since it was introduced from France to the United States in late 1940s. To map the quantitative trait loci (QTL) for stripe rust resistance, an F₈ recombinant inbred line (RIL) population from cross Druchamp × Michigan Amber was phenotyped for stripe rust response in multiple years in fields under natural infection and with selected Pst races under controlled greenhouse conditions, and genotyped with simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. Composite interval mapping (CIM) identified eight HTAP resistance QTL and three all-stage resistance QTL. Among the eight HTAP resistance QTL, QYrdr.wgp-1BL.2 (explaining 2.36-31.04% variation), QYrdr.wgp-2BL (2.81–15.65%), QYrdr.wgp-5AL (2.27–17.22%) and QYrdr.wgp-5BL.2 (2.42–15.13%) were significant in all tests; and QYrdr.wgp-1BL.1 (1.94–10.19%), QYrdr.wgp-1DS (2.04–27.24%), QYrdr.wgp-3AL (1.78–13.85%) and QYrdr.wgp-6BL.2 (1.69–33.71%) were significant in some of the tests. The three all-stage resistance QTL, QYrdr.wgp-5BL.1 (5.47–36.04%), QYrdr.wgp-5DL (9.27–11.94%) and QYrdr.wgp-6BL.1 (13.07-20.36%), were detected based on reactions in the seedlings tested with certain Pst races. Among the eleven QTL detected in Druchamp, at least three (QYrdr.wgp-5DL for race-specific all-stage resistance and QYrdr.wgp-3AL and QYrdr.wgp-6BL.2 for race non-specific HTAP resistance) are new. All these QTL, especially those for durable HTAP resistance, and their closely linked molecular markers could be useful for developing wheat cultivars with durable resistance to stripe rust.