dog chromosome 1 function

Much recent interest in dog genetics has resulted from a desire on the part of veterinary scientists to reduce the problem of inherited diseases in pedigree dogs. human46, mouse47, and gorilla48. Ostrander With these thresholds, we found eight novel genes from the filled CanFam3.1 gaps, and all located in regions with good synteny of human hg38 assembly. Ryder Zheng Association between polymorphisms in the SOX9 region and canine disorder of sex development (78,XX; SRY-negative) revisited in a multibreed case-control study. A. C., Ricketts, S. L., Kuehn, M. H. & Mellersh, C. S. Primary closed angle glaucoma in the Basset Hound: genetic investigations using genome-wide association and RNA sequencing strategies. Fletcher Public microRNA-seq samples (Supplementary Data 1) were combined with the above brain microRNA-seq reads (Total reads, 1.3 billion). Zou, H., Chen, H., Zhou, Z., Wan, Y. Langston Dogs have approximately three billion base pairs in each cell. Dogs come in countless shapes and sizes and exhibit more diversity than any other land mammal on earth, a fact that makes them especially appealing to geneticists. Nash 2D and E). Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen), PCR products and plasmids sequenced using the Mix2Seq service (Eurofins Genomics) and analysed using CodonCode Aligner v6.0.2 (CodonCode). Additional filtering was applied to remove transcripts that, (1) were long single exon transcripts (>10kb and <10% intronic sequence) or (2) originated from genomic polyA/T regions. Hoeppner, M. P. et al. Thomas The genomic architecture of segmental duplications and associated copy number variants in dogs. We noted six tier1 & 2 COSMIC genes that contained either dark or camouflaged regions (EPHA3, RALGDS, LRP1B, CSMD3, ZMYM2, PTEN; 0.86.6% of coding region hidden), potentially masking drivers of disease. 8, 1071610724 (2015). An improved microRNA annotation of the canine genome. North Am. On the other hand, the haploid genome is about 2445 Mb. M Van der Auwera, G. A. et al. BMC Genomics 21, 307 (2020). 2c) and 35 (Supplementary Fig. JM A chromosome is a long, stringy aggregate of genes that carries heredity information and is formed from condensed chromatin. GD Friedlnder, M. R., Mackowiak, S. D., Li, N., Chen, W. & Rajewsky, N. miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Further scaffolding using 94 of 10x and 48 of HiC linked reads resulted in 39 single-scaffold chromosomes (total 2.35Gb) and 2159 unplaced scaffolds (total 128.5Mb; Fig. Chromosomes 1 and 3 are said to be metacentric in homo sapiens. If all the DNA in the cells . Novel origins of copy number variation in the dog genome. Genet. What does cytogenetics do? - Segirt Last Minute Latest News Google Scholar. Now they must determine if the changes that were detected in the genetic code are actually changing the way the gene works. G Honeycutt performed the DNA/RNA extractions. Humans normally have 23 pairs of chromosomes (22 autosomes and 1 sex chromosome), 23 from the mother and 23 from the father. Pharmacogenetics 14, 769773 (2004). The blue indicates a forward alignment and the red indicates a reverse alignment. To identify which chromosome harbored the majority of the DEGs, we analyzed the chromosomal location of all DEGs. Description. Chromosome 1: MedlinePlus Genetics X Chromosome Function & Disorders | What Is the X Chromosome Commun Biol 4, 185 (2021). J Holmes RT C Dogs are used as comparative models for human xenobiotic metabolism, and while a CYP1A2 premature stop codon (rs852922442 C>T) has been reported41,42, the CNV locus expansion has not. Friedrich, J. et al. Transcriptome analysis of dog oral melanoma and its oncogenic analogy Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden, Chao Wang,Ola Wallerman,Maja-Louise Arendt,Elisabeth Sundstrm,sa Karlsson,Jessika Nordin,Suvi Mkelinen,Gerli Rosengren Pielberg,Jennifer R. S. Meadows&Kerstin Lindblad-Toh, Department of Veterinary Clinical Sciences, University of Copenhagen, Frederiksberg D, Denmark, Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden, Suvi Mkelinen,sa Ohlsson&Tomas F. Bergstrm, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden, Jeanette Hanson,Sara Saellstrm,Henrik Rnnberg,Ingrid Ljungvall,Jens Hggstrm&ke Hedhammar, Broad Institute of MIT and Harvard, Cambridge, MA, USA, You can also search for this author in Study Guide Chromosomes and Alleles SP23.pdf - | Course Hero 26, 48864895 (2017). Each cell contains a copy of the mutated gene identical to that from the original mutated cell. A similar analysis was done using 526 dogs from 14 small breeds and nine giant dog breeds. Molin, A.-M., Berglund, J., Webster, M. T. & Lindblad-Toh, K. Genome-wide copy number variant discovery in dogs using the CanineHD genotyping array. Fate Chem. Scaffolding correctness was evaluated by aligning scaffold sequences onto the high-density canine linkage map15. Genome Biol. . We identified 7468 closed CanFam3.1 gaps containing either an exon or promoter sequence as defined by ATAC-seq peaks, accounting for 5743 unique coding exons which were missing in CanFam3.1 (Fig. The diagrams below show stages of mitosis. Introduction To Chromosome 1 - Genetic Education Both fall under the umbrella of National Genomics Infrastructure (NGI) Sweden and Science for Life Laboratory, Sweden and themselves are supported by RFI/VR and the Swedish Research Council and the Knut and Alice Wallenberg Foundation respectively. dog chromosome 1 function Zhong, Z. et al. Assembly and Analysis of Unmapped Genome Sequence Reads Reveal Novel Sequence and Variation in Dogs, Characterisation and functional predictions of canine long non-coding RNAs, Whole genome sequencing of canids reveals genomic regions under selection and variants influencing morphology, Jasmine and Iris: population-scale structural variant comparison and analysis, Construction of JRG (Japanese reference genome) with single-molecule real-time sequencing, Extensive and deep sequencing of the Venter/HuRef genome for developing and benchmarking genome analysis tools, A curated dataset of modern and ancient high-coverage shotgun human genomes, Towards a reference genome that captures global genetic diversity, Highly accurate long-read HiFi sequencing data for five complex genomes, http://hgdownload.soe.ucsc.edu/admin/exe/linux.x86_64.v369/, https://github.com/PapenfussLab/StructuralVariantAnnotation, http://genome-euro.ucsc.edu/cgi-bin/hgTracks?db=canFam4, https://doi.org/10.1101/2020.07.31.231761, https://www.skk.se/sv/Agria-SKK-Forskningsfond/, Description of Additional Supplementary Files, http://creativecommons.org/licenses/by/4.0/, De-novo and genome-wide meta-analyses identify a risk haplotype for congenital sensorineural deafness in Dalmatian dogs, Bayesian model and selection signature analyses reveal risk factors for canine atopic dermatitis, Chromosome-length genome assembly and structural variations of the primal Basenji dog (Canis lupus familiaris) genome, Sign up for Nature Briefing: Translational Research. Long read libraries were prepared with SMRTbell Template Prep Kit 1.0 and 70 SMRT cells were sequenced on the PacBio Sequel system with v2.1 chemistry (Pacific Biosciences; 276.86Gb data). Henthorn B) Each chromosome is replicated into two chromatids during the S phase of interphase. Provided by the Springer Nature SharedIt content-sharing initiative. Different dog breeds developed from small founder populations lollowed by carefully controlled breeding. While the original draft sequence was of good quality, gaps were abundant particularly in promoter regions of the genome, negatively impacting the annotation and study of candidate genes. Sixteen linkage groups of 2 or more markers were identified, and 2 were assigned to defined chromosomes L13 to CFA20 and L 16 to CFA 18. VM Heredity 123, 746758 (2019). answer choices. Hotspots of mutation and breakage in dog and human chromosomes CS GSD_1.0 has a 55-fold increased contiguity compared with its predecessor CanFam3.1. DiGeorge syndrome, more accurately known by a broader term 22q11.2 deletion syndrome is a disorder caused when a small part of chromosome 22 is missing. While we can't tell you exactly what each chromosome does, per se, because one chromosome can have tens of thousands of genes, we can share the chromosome number corresponding to each of our health conditions and traits in their descriptions. chromosome number, precise number of chromosomes typical for a given species. For sequencing coverage, bamCoverage (Deeptools78 v3.3.2) with a 25bp window was used, with unmapped reads and secondary alignments excluded from the analysis. P Transient structural variations have strong effects on quantitative traits and reproductive isolation in fission yeast. Acland Genome 13, 380387 (2002). Bioinformatics 43, 11.10.111.10.33 (2013). USA 106, 27942799 (2009). Scripts used in the study are available at the GitHub repository (https://github.com/Chao912/Mischka/). Awano, T. et al. Applebaum These mutations lead to variant forms of a gene, called alleles, which can change the function of the gene. spanning the relevant 34 million- to 49 million-base-pair interval of chromosome 15 in 353 dogs representing 14 small breeds and 9 giant breeds. 4a, b). 2008; Parker et al. A dog's body contains trillions of cells. After accounting for CYP1A2 SNP rs852922442-T, no significant relative gene expression difference was observed, leaving the phenotypic consequence of this expansion unresolved (CNV 3 vs >3; Supplementary Table7). F Canfam_GSD: de novo chromosome-length genome assembly of the German Shepherd Dog (Canis lupus familiaris) using a combination of long reads, optical mapping, and Hi-C. GigaScience 9,giaa027 (2020). Frontiers | Current Understanding of the Genetics of Intervertebral Visit the Image . dog chromosome 1 function. SNPs and short indels were detected in 10x and ISR dataset using appropriate modules from GATK4. Genetic dissection of complex behaviour traits in German Shepherd dogs. and E.S., C.W., OW, J.R.S.M. Due to the nature of dark and camouflaged regions, default practices will not allow for the mapping of ISR reads to, and subsequent variant extraction from, these positions. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA225755, The Knut and Alice Wallenberg Foundation and Agria och Svenska Kennelklubben Forskningsfond (https://www.skk.se/sv/Agria-SKK-Forskningsfond/, grant numbers: P2012-0015, N2013-0020, P2014-0018, P2015-0012). Acland If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The authors declare no competing interests. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Using new and sophisticated approaches, talented bioinformaticians can compare genome sequence from large numbers of individuals to find single mutations. LV Males have one X and one Y chromosome. Methods 14, 297301 (2017). To make the assembly as complete as possible we therefore combined the output from multiple runs, used TAMA to assemble long reads not overlapping with Stringtie2 transcripts and included novel transcripts if they were multi-exonic and had a blast hit covering at least 50% of the target. In humans, the twenty-third pair is the sex chromosomes, while the first 22 pairs are called autosomes. Marker Name Marker Type Repeat/Gene Name Forward Primer Reverse Primer BP-Size Run T PIC; SEC61G: Death of PRDM9 coincides with stabilization of the recombination landscape in the dog genome. . The sequence of each gene is called its "code." Gu Post author By ; Post date masked singer judges wearing same clothes 2021; drupal is platform dependent true or false . The term "canine genome" refers to the entire sequence of the dog genome including all the genes and the spaces in between. The annotation consisted of 159 thousand transcripts in 29,583 genes; of which 20,654 had an open reading frame (ORF) of at least 100 amino acids and 19,691 genes had a significant BLAST hit against proteins in Swissprot or ENSEMBL. collected the samples with the help of J.H., .O., S.S., H.R., I.L., S.M., J. Hggstrm and .H. High-resolution comparative analysis of great ape genomes. Amorim Improved metagenomic analysis with Kraken 2. a Size distribution and overlap with exons and promoters for the filled CanFam3.1 gaps. PLoS ONE 14, e0218565 (2019). Any window with Fcamouflaged>0.9 was selected as a candidate. Wong, A. K. et al. J. Mol. . Each species has a set number of chromosomes arranged in pairs within each cell, but the number of chromosomes can differ between species. Biol. A chromosome can be defined as an entire chain of DNA and it comes along with a group of stabilizing proteins. Circulating exosomes suppress the induction of regulatory T cells via let-7i in multiple sclerosis. EA Genet. Using HiC and BAC end sequencing data, we confirmed that the inverted GSD_1.0 orientation was correct and refined the placement of regions M1, M2 and M3 (Fig. Chromosomes: Facts about our genetic storerooms | Live Science RL a missense variation in SOD1 leading to degenerative myelopathy5) through complex genomic rearrangements (e.g. Ameur, A. et al. Wayne Rice . & Liu, Z. ATXN3 promotes breast cancer metastasis by deubiquitinating KLF4. Baldwin The term chromosome comes from the Greek . P How Many Chromosomes Do Dogs Have? | Biology Explorer De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds. The family, which now comprises 34 extant species, shows a wide range of chromosome morphologies, with the diploid chromosome number varying from 2n=36 (with mainly metacentric autosomes) in the red fox ( Vulpes vulpes ) to 2n:78 (with all autosomes being acrocentric) in the domestic dog and also a number of wolf-like canids such as the gray wolf ( Canis lupus ). Puck SJ The diploid genes can be classified into 38 pairs with two sex chromosomes. Tengvall, K. et al. These chr 9 insertions are missing from GSD_1.0, but allelic depth analysis revealed that most 10x dogs (26/27) carry between 2 and 6 chr 9 copies (Fig. CS MM Genome Biol. Google Scholar. EA Genetics and Purebred Dogs 101: Part Two - American Kennel Club Putative centromere sequences were annotated if the repeat content was >80%. Ferguson We live in an age that is exploding with knowledge and information. Courtesy of the NHGRI Intramural Publication Support Office. 10). 2006). We present GSD_1.0, a high-quality domestic dog reference genome with chromosome length scaffolds and contiguity increased 55-fold over CanFam3.1. NA Polymorphisms in this gene have previously been associated with psychiatric and neurological disorders (bipolar disorder35 and Alzheimers disease36), and response to associated drug therapies of schizophrenia37 in humans. 3, 9598 (2016). Deep sequencing of a candidate region harboring the SOX9 gene for the canine XX disorder of sex development. Passed from parents to offspring, DNA contains the specific instructions that make each type of living creature unique. Genetics: Dog Coat Color - Principles of Biology This miRNA has been implicated in several human diseases, including multiple sclerosis17, gastric cancer18 and breast cancer19, but has yet to be extensively studied in dogs. In order to locate nucleotide sequences responsible for traits of interest, researchers have to comb through approximately 3 billion base pairs, the length of the canine genome. The histone can be thought of as a spool and the DNA as . SM a GSD_1.0 ideogram showing chromosomes, contigs, gaps, centromere and telomere repeats. Because tetranucleotide repeats have definite advantages over dinucleotide repeats for scoring alleles (generally showing a simpler signal on analysis and being more polymorphic), their use is becoming increasingly popular. Binns What are the functions of Chromosomes? - unacademy.com The majority of the established synteny groups are correlated with linkage groups so that as more of the linkage groups become fixed to chromosomes, gross comparative gene organization in the dog will rapidly become defined. J Chromosomal evolution of the Canidae I: species with high diploid numbers. RK For PacBio, full-length circular consensus sequencing (CCS) reads with at least three passes were selected. The completion of key regions to the investigation of immunological disease and cancer, e.g. We found that the highest number of upregulated genes (n=104) were on CFA 9 (dog chromosome 9) and the highest number of downregulated genes (n=96) were on CFA 1 (dog chromosome 1) (Fig. C) Each chromosome separates into two daughter chromosomes by binary fission. Wayne Ramrez, F. et al. K.L.-T., J.R.S.M. Refinement of the dog map will facilitate the identification of candidate genes for these complex disorders in human and other species through comparative mapping. The availability of a large number of markers will allow the evolutionary relationships between the breeds to be investigated in more detail and should allow breed histories to be established on a more scientific basis than is currently possible. These arms are held together at the center by the centromere. As discussed above, each chromosome is made up of protein and DNA (a single molecule). Notably, eight genes with expression across multiple tissues were completely absent or represented by pseudogenes in CanFam3.1 but were now available for interrogation (PSMA4, CDHR5, SCT, PAOX, UTF1, EFNA2, GPX4 and SLC25A22). Wayne PubMed Central Genetic variation occurs when "mistakes" are made in the cell's duplication or repair mechanisms that cause a permanent change in the nucleotide sequence of the gene. M The commercially available mapping panel comprises 43 microcell hybrid clones containing 1 to 7 canine chromosomes, and 3 whole cell hybrid clones, with each one including 10 to 20 canine chromosomes. Results Next generation . Zajac Work on the dog genome at the Animal Health Trust is generously supported by the Guide Dogs for the Blind Association. b The individual pieces from the reference are plotted as they appear in the alternative haplotig sequence (000151F_042) for Mischka (CNV=3). Because the instinctive behaviors inherited from wild ancestors have been selected to varying degrees in different breeds, certain behavior patterns are now strongly associated with particular breeds. 5, R12 (2004). and .O. A lower cutoff was applied in low-coverage samples to select a maximum of 60Mb (Supplementary Data3). Bowtie alignments of unique sequences were used for MiRDeep271 analysis and compared to known dog and human miRNAs (miRBase) in order to identify the position of both known and novel miRNAs. 11b). Commun. PCR was performed with either PrimeSTAR GXL DNA Polymerase (Takara) or AmpliTaq Gold DNA Polymerase (Applied Biosystems) according to the manufacturers recommendations. Most have nothing to do with disease, but they serve as street signs ("markers") for navigating the dog genome. et al. Several resources have been developed to complement the basic genetic linkage mapping of microsatellite markers that has been carried out on the reference families described above. Drug Metab. New Primers and probes were designed using Primer3 v0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/) and collated in Supplementary Data2. Rare germline variants in known melanoma susceptibility genes in familial melanoma. Correspondence to V Mise, M., Hashizume, T. & Komuro, S. Characterization of substrate specificity of dog CYP1A2 using CYP1A2-deficient and wild-type dog liver microsomes. Behavioral attributes are important characteristics of each dog breed and have been subject to strong selection pressure since the domestication of the dog. PS 30 seconds. and S.M. 10, e1004016 (2014). Want to read all 8 pages? These chromosomes are tightly packed inside the nucleus of a cell and are made of DNA molecules. However, with the help of Bioinformatics, the statistical approach to understanding extensive biological data sets, we have access to many tools for unravelling the secrets of the dog genome. Patterson Domestic dogs have lived alongside humans for at least 10,000 years1,2, and during this time, they have adapted to a shared environment and diet, while being selectively bred for traits such as morphology3 and behaviour4. Chromosomes are located within the nucleus of our cells. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. 2005). Two copies of chromosome 1, one copy inherited from each parent, form one of the pairs. Phased diploid genome assembly with single-molecule real-time sequencing. 3, RESEARCH0034 (2002). Wood, D. E., Lu, J. 9, 17 (2018). Whole genome sequencing of canids reveals genomic regions under selection and variants influencing morphology. Nucleic Acids Res. Binns Cancer 18, 696705 (2018). Females have two X chromosomes. HF Holmes Physical and linkage mapping of human chromosome 17 loci to dog chromosomes 9 and 5, Linkage of a microsatellite marker to the canine copper toxicosis locus in Bedlington terriers, Canine X chromosome-linked hereditary nephritis: A genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the a5 chain of collagen type IV, Institute for Laboratory Animal Research, Laboratory Codes in Nomenclature and Scientific Communication (Advancing Organism Nomenclature in Scientific Communication to Improve Research Reporting and Reproducibility), ILAR: A Retrospective and Prospective Look, History of the National Academies of Sciences, Engineering, and Medicines Institute for Laboratory Animal Research, History of the International Council for Laboratory Animal Science, International Standardized Nomenclature for Outbred Stocks of Laboratory Animals, About the Institute for Laboratory Animal Research, Anticipated Future Contributions of the Map, Receive exclusive offers and updates from Oxford Academic, M. Olivier and M. Breen, personal communication, 1997, Copyright 2023 Institute for Laboratory Animal Research. Reimann dog chromosome 1 function - allinsuranceinfo.org 1a). MG Long-read sequence assembly of the gorilla genome. 36, 19031908 (2008). We compared dog DLA, TRA and TRB regions between GSD_1.0 and CanFam3.1 by NUCMER73. The retina sample was sequenced using both the nanopore direct cDNA sequencing kit SQK-DCS109 and as stranded 2150bp reads on a NovaSeq 6000 S4 lane (Illumina). A canine bacterial artificial chromosome (BAC 1 ) library of approximately 150,000 clones has recently been constructed (the Internet address of Roswell Park Canine BAC Library is provided below). M 20, 117 (2019). Crandall AA RK Blanton The sequence of the dog genome was published in 2005 (Lindblad-Toh et al. PCR fragments were cloned using either Zero Blunt or TOPO TA Cloning Kit (Invitrogen) depending on PCR overhang. An Improved Canine Genome and a Comprehensive Catalogue of - PLOS Preprint at https://arxiv.org/abs/1207.3907 (2012). A novel canine reference genome resolves genomic architecture and uncovers transcript complexity, https://doi.org/10.1038/s42003-021-01698-x. Curr. The individual dark regions were merged, and the dark fraction for each window was assessed for both ISR and 10x datasets: windows with Fdark>0.9 (90% individuals, in at least 23 ISR dogs or 25 10x dogs) retained as the candidate dark regions. Genetics is an area with exponential growth in recent years, and much of this is due to recent technological advances that have allowed for the entire genome of many species, including dogs, to be deciphered. Unplaced GSD_1.0 scaffolds were concatenated into a single scaffold with 500 N base spacers and 10x reads were mapped to each with the Long Ranger v2.2.2 WGS pipeline (10x Genomics). First, total RNA from hypothalamus (RIN>8; Zyagen) was purchased for sequencing via PacBio Iso-Seq express protocol. & Birol, I. ARCS: scaffolding genome drafts with linked reads. . JM Genome Biol. . We mapped Illumina short read libraries from a diverse collection of 118 publically available canid genomes to the Li et al. Note: DCM1 and DCM2 are two separate tests. Proc. With regard to size and weight for example, there is at least a 30-fold difference between the Chihuahua and the Saint Bernard. End pairs that mapped to both assemblies were compared and defined as concordant when they aligned in forward and reverse direction with a distance <500kb. For example, progressive retinal atrophy (PRA 1 ) is equivalent to human retinitis pigmentosa (RP 1 ). Methods 10, 563569 (2013). Genome sequencing highlights the dynamic early history of dogs. They fall in the latter category. Somberg RK Radiation treatment is used as a "local therapy," directed at killing cells within the tumor site itself. Gentleman 07/03/2022 . The black or brown nose correlated perfectly with the absence or presence of the same three TYRP1 variants described above. Different dog breeds therefore present unique opportunities for behavioral genetic studies. RK Reads from the same study and tissue were combined and adaptors were trimmed with BBmap. PS wolf dogs for sale in oklahoma; ms state refund schedule 2022. kde si rychlo pozicat peniaze; can you get crystal serpent in hallowed desert; . BMC Genomics 17, 299 (2016). Humans with Down Syndrome often enjoy a high quality of life. Humans, for instance, have 46 chromosomes in a typical body cell (somatic cell), while dogs have 78 ^1 1. Science 356, 9295 (2017). DLA and TCR, when combined with large reference populations, will facilitate the more accurate genotyping of these regions and hopefully fast track the process from association to causation. Many of the microsatellites derived from the domestic dog are polymorphic in other canids, and indeed they have been used to look at wild canid populations. PJ 19, 13481357 (2017). Somberg CF If a single cell accumulates enough mutations or acquires variation in a critical gene the cell may begin to divide and grow uncontrollably. Slider with three articles shown per slide. Dogs, which are under the species Canis lupus familiaris, are known to have a total of 78 chromosomes (2n). Report on the progress of standardisation of the G-banded canine, Committee for the standardized karyotype of the dog, Multiple and ancient origins of the domestic dog. High molecular weight (HMW) DNA was extracted from blood with MagAttract HMW DNA Kit (Qiagen).

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