Putative Copy Number Variants Research Articles

Uncovering the genetic diversity in Aedes aegypti insecticide resistance genes through global comparative genomics

Aedes aegypti is vector of many arboviruses including Zika, dengue, yellow fever, West Nile, and Chikungunya. Its control efforts are hampered by widespread insecticide resistance reported in the Americas and Asia, while data from Africa is more limited. Here we use publicly available 729 Ae. aegypti whole-genome sequencing samples from 15 countries, including nine in Africa, to investigate the genetic diversity in four insecticide resistance linked genes: ace-1, GSTe2, rdl and vgsc. Apart from vgsc, the other genes have been less investigated in Ae. aegypti, and almost no genetic diversity information is available. Among the four genes, we identified 1,829 genetic variants including 474 non-synonymous substitutions, some of which have been previously documented, as well as putative copy number variations in GSTe2 and vgsc. Global insecticide resistance phenotypic data demonstrated variable resistance in geographic areas with resistant genotypes. Overall, our work provides the first global catalogue and geographic distribution of known and new amino-acid mutations and duplications that can be used to guide the identification of resistance drivers in Ae. aegypti and thereby support monitoring efforts and strategies for vector control.

Putative climate adaptation in American pikas (Ochotona princeps) is associated with copy number variation across environmental gradients

Improved understanding of the genetic basis of adaptation to climate change is necessary for maintaining global biodiversity moving forward. Studies to date have largely focused on sequence variation, yet there is growing evidence that suggests that changes in genome structure may be an even more significant source of adaptive potential. The American pika (Ochotona princeps) is an alpine specialist that shows some evidence of adaptation to climate along elevational gradients, but previous work has been limited to single nucleotide polymorphism based analyses within a fraction of the species range. Here, we investigated the role of copy number variation underlying patterns of local adaptation in the American pika using genome-wide data previously collected across the entire species range. We identified 37–193 putative copy number variants (CNVs) associated with environmental variation (temperature, precipitation, solar radiation) within each of the six major American pika lineages, with patterns of divergence largely following elevational and latitudinal gradients. Genes associated (n = 158) with independent annotations across lineages, variables, and/or CNVs had functions related to mitochondrial structure/function, immune response, hypoxia, olfaction, and DNA repair. Some of these genes have been previously linked to putative high elevation and/or climate adaptation in other species, suggesting they may serve as important targets in future studies.

Accurate and Effective Detection of Recurrent Copy Number Variants in Large SNP Genotype Datasets.

Structural variations, including recurrent Copy Number Variants (CNVs) at specific genomic loci, have been found to be associated with increased risk of several diseases and syndromes. CNV carrier status can be determined in large collections of samples using SNP arrays and, more recently, sequencing data. Although there is some consensus among researchers about the essential steps required in such analysis (i.e., CNV calling, filtering of putative carriers, and visual validation using intensity data plots of the genomic region), standard methodologies and processes to control the quality and consistency of the results are lacking. Here, we present a comprehensive and user-friendly protocol that we have refined from our extensive research experience in the field. We cover every aspect of the analysis, from input data curation to final results. For each step, we highlight which parameters affect the analysis the most and how different settings may lead to different results. We provide a pipeline to run the complete analysis with effective (but customizable) pre-sets. We present software that we developed to better handle and filter putative CNV carriers and perform visual inspection to validate selected candidates. Finally, we describe methods to evaluate the critical sections and actions to counterbalance potential problems. The current implementation is focused on Illumina SNP array data. All the presented software is freely available and provided in a ready-to-use docker container. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: From raw intensity data files to CNV calls Basic Protocol 2: From CNV calls to validated CNV carriers. Basic Protocol 3: Quality control and quality assessment Basic Protocol 4: Install the necessary software.

Accurate in silico confirmation of rare copy number variant calls from exome sequencing data using transfer learning.

Exome sequencing is widely used in genetic studies of human diseases and clinical genetic diagnosis. Accurate detection of copy number variants (CNVs) is important to fully utilize exome sequencing data. However, exome data are noisy. None of the existing methods alone can achieve both high precision and recall rate. A common practice is to perform heuristic filtration followed by manual inspection of read depth of putative CNVs. This approach does not scale in large studies. To address this issue, we developed a transfer learning method, CNV-espresso, for in silico confirming rare CNVs from exome sequencing data. CNV-espresso encodes candidate CNVs from exome data as images and uses pretrained convolutional neural network models to classify copy number states. We trained CNV-espresso using an offspring-parents trio exome sequencing dataset, with inherited CNVs as positives and CNVs with Mendelian errors as negatives. We evaluated the performance using additional samples that have both exome and whole-genome sequencing (WGS) data. Assuming the CNVs detected from WGS data as a proxy of ground truth, CNV-espresso significantly improves precision while keeping recall almost intact, especially for CNVs that span a small number of exons. CNV-espresso can effectively replace manual inspection of CNVs in large-scale exome sequencing studies.

Genome-wide elucidation of CNV regions and their association with production and reproduction traits in composite Vrindavani cattle

The present study was aimed to analyze the genome-wide copy number variations (CNVs) in Vrindavani composite cattle and concatenate them into CNV regions (CNVRs), and finally test the association of CNVRs with different production and reproduction traits. Genotypic data, generated on BovineSNP50 Beadchip (v3) array for 96 Vrindavani animals, was used to elucidate the CNVs at the genome level. Intensity data covering over 53,218 SNP genotypes on bovine genome was used. Algorithm based on Hidden Markov Model was employed in PennCNV program to detect, normalize and filter CNVs across the genome. 252 putative CNVs, detected via PennCNV program, in different individuals were concatenated into 71 CNV regions (CNVRs) using CNVRuler program. Association of CNVRs with important (re)production traits in Vrindavani animals was assessed using linear regression. Five CNVRs were found to be significantly associated with ten important (re)production traits. The genes harbored in these regions provided useful insights into the association of CNVRs with genes and ultimately the variation at phenotype level. Important genes that overlapped with CNVRs included WASHC4, HS6ST3, MBNL2, TOLLIP, PIDD1 and TSPAN4. Furthermore, the CNVRs were found to overlap with important QTLs available in AnimalQTL database which affect milk yield and composition along with reproduction and immune function traits. The copy number states of three enes were validated using digital droplet PCR technique. The results from the present study significantly enhance the understanding about CNVs in Vrindavani cattle and should help establish its CNV map. The study will also enable further investigation on association of these variants with important traits of economic interest including disease incidence.

Genomic basis of deep-water adaptation in Arctic Charr (Salvelinus alpinus) morphs.

The post-glacial colonization of Gander Lake in Newfoundland, Canada, by Arctic Charr (Salvelinus alpinus) provides the opportunity to study the genomic basis of adaptation to extreme deep-water environments. Colonization of deep-water (>50m) habitats often requires extensive adaptation to cope with novel environmental challenges from high hydrostatic pressure, low temperature, and low light, but the genomic mechanisms underlying evolution in these environments are rarely known. Here, we compare genomic divergence between a deep-water morph adapted to depths of up to 288m and a larger, piscivorous pelagic morph occupying shallower depths. Using both a SNP array and resequencing of whole nuclear and mitochondrial genomes, we find clear genetic divergence (FST =0.11-0.15) between deep and shallow water morphs, despite an absence of morph divergence across the mitochondrial genome. Outlier analyses identified many diverged genomic regions containing genes enriched for processes such as gene expression and DNA repair, cardiac function, and membrane transport. Detection of putative copy number variants (CNVs) uncovered 385genes with CNVs distinct to piscivorous morphs, and 275genes with CNVs distinct to deep-water morphs, enriched for processes associated with synapse assembly. Demographic analyses identified evidence for recent and local morph divergence, and ongoing reductions in diversity consistent with postglacial colonization. Together, these results show that Arctic Charr morph divergence has occurred through genome-wide differentiation and elevated divergence of genes underlying multiple cellular and physiological processes, providing insight into the genomic basis of adaptation in a deep-water habitat following postglacial recolonization.

Genomic signatures of thermal adaptation are associated with clinal shifts of life history in a broadly distributed frog.

Temperature is a critical driver of ectotherm life‐history strategies, whereby a warmer environment is associated with increased growth, reduced longevity and accelerated senescence. Increasing evidence indicates that thermal adaptation may underlie such life‐history shifts in wild populations. Single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) can help uncover the molecular mechanisms of temperature‐driven variation in growth, longevity and senescence. However, our understanding of these mechanisms is still limited, which reduces our ability to predict the response of non‐model ectotherms to global temperature change.In this study, we examined the potential role of thermal adaptation in clinal shifts of life‐history traits (i.e. life span, senescence rate and recruitment) in the Columbia spotted frog Rana luteiventris along a broad temperature gradient in the western United States.We took advantage of extensive capture–recapture datasets of 20,033 marked individuals from eight populations surveyed annually for 14–18 years to examine how mean annual temperature and precipitation influenced demographic parameters (i.e. adult survival, life span, senescence rate, recruitment and population growth). After showing that temperature was the main climatic predictor influencing demography, we used RAD‐seq data (50,829 SNPs and 6,599 putative CNVs) generated for 352 individuals from 31 breeding sites to identify the genomic signatures of thermal adaptation.Our results showed that temperature was negatively associated with annual adult survival and reproductive life span and positively associated with senescence rate. By contrast, recruitment increased with temperature, promoting the long‐term viability of most populations. These temperature‐dependent demographic changes were associated with strong genomic signatures of thermal adaptation. We identified 148 SNP candidates associated with temperature including three SNPs located within protein‐coding genes regulating resistance to cold and hypoxia, immunity and reproduction in ranids. We also identified 39 CNV candidates (including within 38 transposable elements) for which normalized read depth was associated with temperature.Our study indicates that both SNPs and structural variants are associated with temperature and could eventually be found to play a functional role in clinal shifts in senescence rate and life‐history strategies in R. luteiventris. These results highlight the potential role of different sources of molecular variation in the response of ectotherms to environmental temperature variation in the context of global warming.

Thermal adaptation rather than demographic history drives genetic structure inferred by copy number variants in a marine fish

Increasing evidence shows that structural variants represent an overlooked aspect of genetic variation with consequential evolutionary roles. Among those, copy number variants (CNVs), including duplicated genomic region and transposable elements (TEs) may contribute to local adaptation and/or reproductive isolation among divergent populations. Those mechanisms suppose that CNVs could be used to infer neutral and/or adaptive population genetic structure, whose study has been restricted to microsatellites, mtDNA, and AFLP markers in the past and more recently the use of SNPs. Taking advantage of recent developments allowing CNV analysis from RAD-seq data, we investigated how variation in fitness-related traits, local environmental conditions and demographic history are associated with CNVs, and how subsequent copy number variation drives population genetic structure in a marine fish, the capelin (Mallotus villosus). We collected 1538 DNA samples from 35 sampling sites in the north Atlantic Ocean and identified 6620 putative CNVs. We found associations between CNVs and the gonadosomatic index, suggesting that six duplicated regions could affect female fitness by modulating oocyte production. We also detected 105 CNV candidates associated with water temperature, among which 20% corresponded to genomic regions located within the sequence of protein-coding genes, suggesting local adaptation to cold water by means of gene sequence amplification. We also identified 175 CNVs associated with the divergence of three previously defined parapatric glacial lineages, of which 24% were located within protein-coding genes, making those loci potential candidates for reproductive isolation. Lastly, our analyses unveiled a hierarchical, complex CNV population structure determined by temperature and local geography, which was in stark contrast with that inferred based on SNPs in a previous study. Our findings underscore the complementarity of those two types of genomic variation in population genomics studies.

SeeCiTe: a method to assess CNV calls from SNP arrays using trio data.

MotivationSingle nucleotide polymorphism (SNP) genotyping arrays remain an attractive platform for assaying copy number variants (CNVs) in large population-wide cohorts. However, current tools for calling CNVs are still prone to extensive false positive calls when applied to biobank scale arrays. Moreover, there is a lack of methods exploiting cohorts with trios available (e.g. nuclear family) to assist in quality control and downstream analyses following the calling.ResultsWe developed SeeCiTe (Seeing CNVs in Trios), a novel CNV-quality control tool that postprocesses output from current CNV-calling tools exploiting child-parent trio data to classify calls in quality categories and provide a set of visualizations for each putative CNV call in the offspring. We apply it to the Norwegian Mother, Father and Child Cohort Study (MoBa) and show that SeeCiTe improves the specificity and sensitivity compared to the common empiric filtering strategies. To our knowledge, it is the first tool that utilizes probe-level CNV data in trios (and singletons) to systematically highlight potential artifacts and visualize signal intensities in a streamlined fashion suitable for biobank scale studies.Availability and implementationThe software is implemented in R with the source code freely available at https://github.com/aksenia/SeeCiTeSupplementary information Supplementary data are available at Bioinformatics online.

RBV: Read balance validator, a tool for prioritising copy number variations in germline conditions

The popularisation and decreased cost of genome resequencing has resulted in an increased use in molecular diagnostics. While there are a number of established and high quality bioinfomatic tools for identifying small genetic variants including single nucleotide variants and indels, currently there is no established standard for the detection of copy number variants (CNVs) from sequence data. The requirement for CNV detection from high throughput sequencing has resulted in the development of a large number of software packages. These tools typically utilise the sequence data characteristics: read depth, split reads, read pairs, and assembly-based techniques. However, the additional source of information from read balance (defined as relative proportion of reads of each allele at each position) has been underutilised in the existing applications. Here we present Read Balance Validator (RBV), a bioinformatic tool that uses read balance for prioritisation and validation of putative CNVs. The software simultaneously interrogates nominated regions for the presence of deletions or multiplications, and can differentiate larger CNVs from diploid regions. Additionally, the utility of RBV to test for inheritance of CNVs is demonstrated in this report. RBV is a CNV validation and prioritisation bioinformatic tool for both genome and exome sequencing available as a python package from https://github.com/whitneywhitford/RBV.

Identification of copy number variations using high density whole-genome SNP markers in Chinese Dongxiang spotted pigs.

ObjectiveCopy number variations (CNVs) are a major source of genetic diversity complementary to single nucleotide polymorphism (SNP) in animals. The aim of the study was to perform a comprehensive genomic analysis of CNVs based on high density whole-genome SNP markers in Chinese Dongxiang spotted pigs.MethodsWe used customized Affymetrix Axiom Pig1.4M array plates containing 1.4 million SNPs and the PennCNV algorithm to identify porcine CNVs on autosomes in Chinese Dongxiang spotted pigs. Then, the next generation sequence data was used to confirm the detected CNVs. Next, functional analysis was performed for gene contents in copy number variation regions (CNVRs). In addition, we compared the identified CNVRs with those reported ones and quantitative trait loci (QTL) in the pig QTL database.ResultsWe identified 871 putative CNVs belonging to 2,221 CNVRs on 17 autosomes. We further discarded CNVRs that were detected only in one individual, leaving us 166 CNVRs in total. The 166 CNVRs ranged from 2.89 kb to 617.53 kb with a mean value of 93.65 kb and a genome coverage of 15.55 Mb, corresponding to 0.58% of the pig genome. A total of 119 (71.69%) of the identified CNVRs were confirmed by next generation sequence data. Moreover, functional annotation showed that these CNVRs are involved in a variety of molecular functions. More than half (56.63%) of the CNVRs (n = 94) have been reported in previous studies, while 72 CNVRs are reported for the first time. In addition, 162 (97.59%) CNVRs were found to overlap with 2,765 previously reported QTLs affecting 378 phenotypic traits.ConclusionThe findings improve the catalog of pig CNVs and provide insights and novel molecular markers for further genetic analyses of Chinese indigenous pigs.

Diversity of copy number variation in the worldwide goat population.

Goats (Capra hircus) are an important farm animal species. Copy number variation (CNV) represents a major source of genomic structural variation. We investigated the diversity of CNV distribution in goats using CaprineSNP50 genotyping data generated by the ADAPTmap Project. We identified 6286 putative CNVs in 1023 samples from 50 goat breeds using PennCNV. These CNVs were merged into 978 CNV regions, spanning ~262 Mb of total length and corresponding to ~8.96% of the goat genome. We then divided the samples into six subgroups per geographic distribution and constructed a comparative CNV map. Our results revealed a population differentiation in CNV across different geographical areas, including Western Asia, Eastern Mediterranean, Alpine & Northern Europe, Madagascar, Northwestern Africa, and Southeastern Africa groups. The results of a cluster heatmap analysis based on the CNV count per individual across different groups was generally consistent with the one generated from the SNP data, likely reflecting the population history of different goat breeds. We sought to determine the gene content of these CNV events and found several important CNV-overlapping genes (e.g. EDNRA, ADAMTS20, ASIP, KDM5B, ADAM8, DGAT1, CHRNB1, CLCN7, and EXOSC4), which are involved in local adaptations such as coat color, muscle development, metabolic processes, osteopetrosis, and embryonic development. Therefore, this research generated an extensive CNV map in the worldwide population of goat, which offers novel insight into the goat genome and its functional annotation.

An Extended Targeted Copy Number Variation Detection Array Including 187 Genes for the Diagnostics of Neuromuscular Disorders.

Background:Our previous array, the Comparative Genomic Hybridisation design (CGH-array) for nemaline myopathy (NM), named the NM-CGH array, revealed pathogenic copy number variation (CNV) in the genes for nebulin (NEB) and tropomyosin 3 (TPM3), as well as recurrent CNVs in the segmental duplication (SD), i.e. triplicate, region of NEB (TRI, exons 82–89, 90–97, 98–105). In the light of this knowledge, we have designed and validated an extended CGH array, which includes a selection of 187 genes known to cause neuromuscular disorders (NMDs).Objective:Our aim was to develop a reliable method for CNV detection in genes related to neuromuscular disorders for routine mutation detection and analysis, as a much-needed complement to sequencing methods.Methods:We have developed a novel custom-made 4×180 k CGH array for the diagnostics of NMDs. It includes the same tiled ultra-high density coverage of the 12 known or putative NM genes as our 8×60 k NM-CGH-array but also comprises a selection of 175 additional genes associated with NMDs, including titin (TTN), at a high to very high coverage. The genes were divided into three coverage groups according to known and potential pathogenicity in neuromuscular disorders.Results:The array detected known and putative CNVs in all three gene coverage groups, including the repetitive regions of NEB and TTN.Conclusions:The targeted neuromuscular disorder 4×180 k array-CGH (NMD-CGH-array v1.0) design allows CNV detection for a broader spectrum of neuromuscular disorders at a high resolution.

Diversity of copy number variation in a worldwide population of sheep

Copy number variation (CNV) represents a major source of genomic variation. We investigated the diversity of CNV distribution using SNP array data collected from a comprehensive collection of geographically dispersed sheep breeds. We identified 24,558 putative CNVs, which can be merged into 619 CNV regions, spanning 197Mb of total length and corresponding to ~6.9% of the sheep genome. Our results reveal a population differentiation in CNV between different geographical areas, including Africa, America, Asia, Southwestern Asia, Central Europe, Northern Europe and Southwestern Europe. We observed clear distinctions in CNV prevalence between diverse groups, possibly reflecting the population history of different sheep breeds. We sought to determine the gene content of CNV, and found several important CNV-overlapping genes (BTG3, PTGS1 and PSPH) which were involved in fetal muscle development, prostaglandin (PG) synthesis, and bone color. Our study generates a comprehensive CNV map, which may contribute to genome annotation in sheep.

Evaluating whole genome sequence data from the Genetic Absence Epilepsy Rat from Strasbourg and its related non-epileptic strain.

ObjectiveThe Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbreed Wistar rat strain widely used as a model of genetic generalised epilepsy with absence seizures. As in humans, the genetic architecture that results in genetic generalized epilepsy in GAERS is poorly understood. Here we present the strain-specific variants found among the epileptic GAERS and their related Non-Epileptic Control (NEC) strain. The GAERS and NEC represent a powerful opportunity to identify neurobiological factors that are associated with the genetic generalised epilepsy phenotype.MethodsWe performed whole genome sequencing on adult epileptic GAERS and adult NEC rats, a strain derived from the same original Wistar colony. We also generated whole genome sequencing on four double-crossed (GAERS with NEC) F2 selected for high-seizing (n = 2) and non-seizing (n = 2) phenotypes.ResultsSpecific to the GAERS genome, we identified 1.12 million single nucleotide variants, 296.5K short insertion-deletions, and 354 putative copy number variants that result in complete or partial loss/duplication of 41 genes. Of the GAERS-specific variants that met high quality criteria, 25 are annotated as stop codon gain/loss, 56 as putative essential splice sites, and 56 indels are predicted to result in a frameshift. Subsequent screening against the two F2 progeny sequenced for having the highest and two F2 progeny for having the lowest seizure burden identified only the selected Cacna1h GAERS-private protein-coding variant as exclusively co-segregating with the two high-seizing F2 rats.SignificanceThis study highlights an approach for using whole genome sequencing to narrow down to a manageable candidate list of genetic variants in a complex genetic epilepsy animal model, and suggests utility of this sequencing design to investigate other spontaneously occurring animal models of human disease.

Abstract MIP-075: SOMATIC MUTATIONS AND COPY-NUMBER VARIATIONS IN OVARIAN CANCERS VIA TARGETED SEQUENCING OF EXONS OF 300 CANCER-ASSOCIATED GENES, INCLUDING MULTIPLE DNA DAMAGE REPAIR GENES

Abstract INTRODUCTION: Recent studies demonstrated frequent alterations in DNA damage repair (DDR) genes in ovarian cancer, from well-established variants in BRCA1/2 to rarely altered genes in various DDR pathways that may be clinically relevant. Here we summarize somatic alterations in DDR related genes identified by targeted sequencing of several hundred ovarian cancers. EXPERIMENTAL PROCEDURES: Targeted next-generation sequencing was performed on formalin-fixed tumor tissues from >500 patients with ovarian cancer enrolled in an IRB-approved protocol at Dana-Farber Cancer Institute (DFCI). The current dataset includes 269 patients whose tumors underwent gynecologic pathology review. Targeted sequencing via hybrid capture was performed for exons of 300 cancer-associated genes. Sequence data was analyzed at Brigham and Women's Hospital to identify single-nucleotide variants (SNVs) and insertions/deletions (indels). Putative copy number variants (CNVs) were computationally inferred from sequencing coverage in a subset of patients (n=219). Germline analysis was not performed in this study, but SNVs were filtered against variant databases. Corresponding clinical data is undergoing analysis. RESULTS: In the 269-patient cohort, 67% of samples were primary tumors, 25% metastatic recurrences, and 3% local recurrences. Histologies included high-grade serous (34%), endometrioid (12%), clear cell (9%), malignant mixed mesodermal tumor (MMMT, carcinosarcoma) (9%), low-grade serous (7%), granulosa cell (7%), mucinous (3%), and borderline variants (6%). We focused upon alterations in DDR related genes in the targeted panel. Of the 269 patients, ~9% harbored BRCA1 variants and ~13% harbored BRCA2 variants; frameshift and nonsense mutations were frequent. Copy losses in BRCA1 or BRCA2 were predicted in up to 1/3 of patients, some of whom also carried SNVs/indels in the same gene. BRCA1/2 alterations were more frequent in high-grade serous cancers, but were also observed in other histologic subtypes. SNVs/indels in ATM were common, present in ~9% of patients. Another ~10% of patients had a variant in one of the Fanconi Anemia (FA) genes (FANCA,B,C,D2,E,F,G,I) or BRIP1. Alterations in MMR genes (MLH1, MSH2, MSH6, PMS2) were present in ~12%. SNVs in genes encoding DDR associated helicases BLM or WRN were observed in ~8%. PRKDC, encoding the DNA-PK catalytic subunit involved in non-homologous end joining, was altered in up to 7%. The SNVs in these DDR genes were observed in all histologic subtypes at varying frequencies. CNVs in DDR related genes were common, including predicted amplifications of CCNE1 and 2-copy deletions in several FA genes. Several limitations of this dataset may result in overestimates of the frequency of clinically significant alterations, e.g. some SNVs may represent benign variants (specific mutations are undergoing more detailed analysis to eliminate these); some patients harbor multiple mutations; and CNVs are predicted but need to be confirmed by orthogonal methods. CONCLUSIONS: Targeted sequencing data from a large cohort of ovarian cancer patients collectively support previous work indicating a significant frequency of alterations in DDR related genes in ovarian cancer, across multiple histologic subtypes. Further analysis of specific mutations and clinical correlations with DDR gene alterations are ongoing. Identifying alterations in DDR genes in ovarian cancer patients is feasible and may have clinical relevance, particularly regarding DNA repair targeted agents and chemotherapeutics. Citation Format: Elizabeth Stover, Brooke Howitt, Levi Garraway, Ursula Matulonis. SOMATIC MUTATIONS AND COPY-NUMBER VARIATIONS IN OVARIAN CANCERS VIA TARGETED SEQUENCING OF EXONS OF 300 CANCER-ASSOCIATED GENES, INCLUDING MULTIPLE DNA DAMAGE REPAIR GENES [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-075.

Detection and assessment of copy number variation using PacBio long-read and Illumina sequencing in New Zealand dairy cattle

Single nucleotide polymorphisms have been the DNA variant of choice for genomic prediction, largely because of the ease of single nucleotide polymorphism genotype collection. In contrast, structural variants (SV), which include copy number variants (CNV), translocations, insertions, and inversions, have eluded easy detection and characterization, particularly in nonhuman species. However, evidence increasingly shows that SV not only contribute a substantial proportion of genetic variation but also have significant influence on phenotypes. Here we present the discovery of CNV in a prominent New Zealand dairy bull using long-read PacBio (Pacific Biosciences, Menlo Park, CA) sequencing technology and the Sniffles SV discovery tool (version 0.0.1; https://github.com/fritzsedlazeck/Sniffles). The CNV identified from long reads were compared with CNV discovered in the same bull from Illumina sequencing using CNVnator (read depth-based tool; Illumina Inc., San Diego, CA) as a means of validation. Subsequently, further validation was undertaken using whole-genome Illumina sequencing of 556 cattle representing the wider New Zealand dairy cattle population. Very limited overlap was observed in CNV discovered from the 2 sequencing platforms, in part because of the differences in size of CNV detected. Only a few CNV were therefore able to be validated using this approach. However, the ability to use CNVnator to genotype the 557 cattle for copy number across all regions identified as putative CNV allowed a genome-wide assessment of transmission level of copy number based on pedigree. The more highly transmissible a putative CNV region was observed to be, the more likely the distribution of copy number was multimodal across the 557 sequenced animals. Furthermore, visual assessment of highly transmissible CNV regions provided evidence supporting the presence of CNV across the sequenced animals. This transmission-based approach was able to confirm a subset of CNV that segregates in the New Zealand dairy cattle population. Genome-wide identification and validation of CNV is an important step toward their inclusion in genomic selection strategies.

AB015. The relationship between copy number variations and high myopia in Chinese: a case-control study

: As a complex disease, myopia is the most common eye disease worldwide. Many myopia susceptibility genes or variants have been successfully identified in the past years by genome-wide genetic association studies (GWAS), which focus mainly on the single-nucleotide polymorphisms. Little attention has been paid to examine the role of copy number variations (CNVs) in refractive error and myopia. This study adopted a systematic strategy to investigate the role of CNVs in high myopia. In the discovery phase, a pilot GWAS suggests putative CNVs for follow-up. Multiplex ligation-dependent probe amplification was then used to quantify the copy number of 89 CNV segments in 737 case-control samples in the second phase and then 24 top-ranking CNVs in a second group of 1,029 case-control samples in the final validation phase. This validation phase identified 22 significant CNVs. Further work is needed to examine the role of these few CNVs in myopia development.

Copy number variations in Hanwoo and Yanbian cattle genomes using the massively parallel sequencing data

Hanwoo is an indigenous Korean beef cattle breed, and it shared an ancestor with Yanbian cattle that are found in the Northeast provinces in China until the last century. During recent decades, those cattle breeds experienced different selection pressures. Here, we present genome-wide copy number variations (CNVs) by comparing Hanwoo and Yanbian cattle sequencing data. We used ~3.12 and ~3.07 billion sequence reads from Hanwoo and Yanbian cattle, respectively. A total of 901 putative CNV regions (CNVRs) were identified throughout the genome, representing 5,513,340bp. This is a smaller number than has been reported in previous studies, indicating that Hanwoo are genetically close to Yanbian cattle. Of the CNVRs, 53.2% and 46.8% were found to be gains and losses in Hanwoo. Potential functional roles of each CNVR were assessed by annotating all CNVRs and gene ontology (GO) enrichment analysis. We found that 278 CNVRs overlapped with cattle gene-sets (genic-CNVRs) that could be promising candidates to account for economically important traits in cattle. The enrichment analysis indicated that genes were significantly over-represented in GO terms, including developmental process, multicellular organismal process, reproduction, and response to stimulus. These results provide a valuable genomic resource for determining how CNVs are associated with cattle traits.

Genome-wide CNV analysis in 221 unrelated patients and targeted high-throughput sequencing reveal novel causative candidate genes for colorectal adenomatous polyposis.

To uncover novel causative genes in patients with unexplained adenomatous polyposis, a model disease for colorectal cancer, we performed a genome-wide analysis of germline copy number variants (CNV) in a large, well characterized APC and MUTYH mutation negative patient cohort followed by a targeted next generation sequencing (NGS) approach. Genomic DNA from 221 unrelated German patients was genotyped on high-resolution SNP arrays. Putative CNVs were filtered according to stringent criteria, compared with those of 531 population-based German controls, and validated by qPCR. Candidate genes were prioritized using in silico, expression, and segregation analyses, data mining and enrichment analyses of genes and pathways. In 27% of the 221 unrelated patients, a total of 77 protein coding genes displayed rare, nonrecurrent, germline CNVs. The set included 26 candidates with molecular and cellular functions related to tumorigenesis. Targeted high-throughput sequencing found truncating point mutations in 12% (10/77) of the prioritized genes. No clear evidence was found for autosomal recessive subtypes. Six patients had potentially causative mutations in more than one of the 26 genes. Combined with data from recent studies of early-onset colorectal and breast cancer, recurrent potential loss-of-function alterations were detected in CNTN6, FOCAD (KIAA1797), HSPH1, KIF26B, MCM3AP, YBEY and in three genes from the ARHGAP family. In the canonical Wnt pathway oncogene CTNNB1 (β-catenin), two potential gain-of-function mutations were found. In conclusion, the present study identified a group of rarely affected genes which are likely to predispose to colorectal adenoma formation and confirmed previously published candidates for tumor predisposition as etiologically relevant.