For environmental health research, this means that healthy laboratory zebrafish strains that are sufficiently outbred, and thus of comparable genetic diversity versus other natural populations, can be a powerful model for environmental exposure studies in humans and other species. Finally, we explored whether the higher apparent diversity observed in our T5D line could be due to experimental design factors that tend to underestimate diversity in other published lines. Indeed, the zebrafish model is gaining tractability as a human disease model (Howe et al. We establish T5D as a model that is representative of diversity levels within laboratory zebrafish lines and demonstrate that experimental design and analysis can exert major effects when characterizing genetic diversity in heterogeneous populations. Alignments were further deduplicated using ‘samtools rmdup’. https://doi.org/10.1007/s00335-018-9735-x, DOI: https://doi.org/10.1007/s00335-018-9735-x, Over 10 million scientific documents at your fingertips, Not logged in https://doi.org/10.1007/s00335-012-9414-2, Cirelli C, Tononi G, Mackay TF et al (2008) Is sleep essential? Nat Commun 7:13637. https://doi.org/10.1038/ncomms13637, Churchill GA, Airey DC, Allayee H et al (2004) The collaborative cross, a community resource for the genetic analysis of complex traits. A VCF file for NHGRI-1 (LaFave et al. PLoS Biol 6:e216. The sequencing data are described in detail in (Balik-Meisner et al., submitted). GC content for each sample was ~ 37%, which is consistent with the zebrafish genome (Han and Zhao 2008). First, 20 individuals were randomly selected. The estimate of 20.1 M SNPs segregating in the population (10.3 M in non-repetitive regions of the genome used for zebrafish line comparisons) included non-reference allele frequencies from 0.1 to 99.8%. Thus, this model could be used for large-scale studies of chemical bioactivity that include genetic information on response mechanisms during development of exposed individuals (Baer et al. The team identified 154 pseudogenes in the zebrafish genome, a fraction of the 13,000 or so pseudogenes found in the human genome. https://doi.org/10.1101/gr.107524.110, McLaren W, Gil L, Hunt SE et al (2016) The ensembl variant effect predictor. Continued work on identifying genetic variation in commonly used zebrafish lines will be important for exploration of gene–environment interactions (G×E), epigenetic modifications, and other genetic effects linked to environmental exposure-associated hazards. The zebrafish and the mouse are the most commonly studied vertebrate laboratory animals whose genomes have been completely sequenced. Genotypes are reported for every individual at every variant site for which they had any remaining reads. 2014; Asharani et al. 2014; Butler et al. Approximately 45 M single nucleotide polymorphisms (SNPs) segregate in the CC and DO populations, four times more than in any singular laboratory mouse strain (Yang et al. Many of these sites may actually be variable in the populations (rather than fixed) yet missed in the sampled subsets. This would be consistent with the continued rare variant discovery in human populations noted in the previous section. PubMed Central  J Fish Biol 15:323–327, Nasiadka A, Clark MD (2012) Zebrafish breeding in the laboratory environment. J Occup Environ Hyg 12(Suppl 1):S55–S68. 2015). BMC Genom. For indels, the count decreased from 2,966,260 to 2,608,746 to 2,339,775. 1). Google Scholar, Patowary A, Purkanti R, Singh M et al (2013) A sequence-based variation map of zebrafish. Variants discovered in the other lines in pooled sequencing experiments were primarily common, because a given site had low coverage (< 20×) across the pool. https://doi.org/10.1093/toxsci/kft235, Unckless RL, Rottschaefer SM, Lazzaro BP (2015) A genome-wide association study for nutritional indices in Drosophila. Environ Sci Technol 44:5979–5985. So from a single cell the day they're born, they will have a head, and a tail, and a beating heart within 24 hours. In order to create a RIL panel representing the genetic diversity among a more general populace of mice, the collaborative cross (CC) (Chesler et al. This may be partially explained by the lower coverage per individual in our design, wherein we sacrificed sequencing depth per individual in order to include a larger sample and better estimate genotype frequencies for rare variants. Samples were sheared to ~ 320 bp, and 100 ng was used in the WaferGen robotic DNA library prep. In zebrafish, inbreeding adversely affects fecundity and survival (Mrakovcic and Haley 1979), so endeavors to create isogenic lines have not been fruitful. Toxicol Sci 137:212–233. 2009). The 20.1 M SNPs equate to 13.4 SNPs per 1 kb genomic sequence. Nat Commun. Commonly used to understand gene function. In this regard, zebrafish are useful because the embryo is transparent, it develops outside of its mother, and its development from eggs to larvae happens in just three days. We have generated lesions ranging from small indels to full gene deletions. 2012) or sample size of 2. https://doi.org/10.1038/nmeth.1923, Li H, Handsaker B, Wysoker A et al (2009) The sequence alignment/map format and SAMtools. Changes in genotype frequencies within the population can be tracked, which can address whether genetic drift or unwanted selection is affecting a laboratory population aiming to maintain an “outbred” strategy that maintains diversity. A major difference from many model organisms is that standard husbandry practices in zebrafish are designed to maintain population diversity. The maximum intron size found in each genome is presented in table 1. 2009). As a vertebrate with one of the largest sets of protein-coding genes, consisting of orthologues for over 70% of human genes, they have been adapted as exposure and human disease models (Howe et al. After applying the filtering cutoffs, 20,385,817 SNPs and 6,304,066 indels remained. 2013). Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Nature 482:173–178. https://doi.org/10.3109/17435390.2010.489207, Baer CE, Ippolito DL, Hussainzada N et al (2014) Genome-wide gene expression profiling of acute metal exposures in male zebrafish. 2017). https://doi.org/10.1093/gerona/glv047, Judson RS, Martin MT, Reif DM, Houck KA, Knudsen TB, Rotroff DM et al (2010) Analysis of eight oil spill dispersants using rapid, in vitro tests for endocrine and other biological activity. Model organisms have long been utilized to study genetic determinants underlying human disease susceptibility, because experiments can exert necessary controls over factors such as diet, lifestyle, and environment that would be impossible in a human setting. c Number of models per disease category stacked by organism (from https://monarchinitiative.org). https://doi.org/10.1021/es102150z, Kang L, Aggarwal DD, Rashkovetsky E et al (2016) Rapid genomic changes in Drosophila melanogaster adapting to desiccation stress in an experimental evolution system. 1a). https://doi.org/10.1038/351652a0, Ka-Shu Wong G, Liu B, Wang J et al (2004) A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms. Google Scholar, Bai W, Zhang Z, Tian W et al (2009) Toxicity of zinc oxide nanoparticles to zebrafish embryo: a physicochemical study of toxicity mechanism. Approximately 51% of the genome is masked for having highly repetitive content. https://doi.org/10.1007/s11051-009-9740-9, Article  https://doi.org/10.1002/aja.1002030302, Knecht AL, Truong L, Marvel SW et al (2017) Transgenerational inheritance of neurobehavioral and physiological deficits from developmental exposure to benzo[a]pyrene in zebrafish. While more variants have been discovered in the human and mouse genomes, the smaller zebrafish genome is on par with—or in some cases may even exceed—genetic variability observed between individuals in those species. Size of genome Value 1.41e+9 bp Range: 26,206 protein coding genes bp To date, the total number of discovered variants in the zebrafish genome is less than half the number found in human or mouse genomes; consequently, validation is more sparse. T5D variants, discovered based on approximately 1380× coverage across individuals (5× for 276 individuals), followed an allele frequency spectrum more similar to known human variants (Figs. Nat Methods 9:357–359. Google Scholar, LaFave MC, Varshney GK, Vemulapalli M et al (2014) A defined zebrafish line for high-throughput genetics and genomics: NHGRI-1. After masking variants located in non-complex regions of the genome, the final pooled approximation T5D comparison dataset resulted in 6,175,287 SNPs and 1,080,749 indels. Information about the continuing improvement of the zebrafish genome After 2.5 years of assembly curation, the GRC presents the new zebrafish reference genome assembly, GRCz11. To address the impact of sequence design on comparisons between T5D and other lines that used pooled sequencing, a portion of the T5D data was used as a simulated pool. The zebrafish reference genome sequence and its relationship to the human genome. 2011). When both larvae are diploid ( Fig 1A ), there are two main peaks; in addition to the G1 phase cells there is a second peak showing G2 phase cells with a 4n DNA content. The table includes a link to the talks and manuals from the 2018 workshop for further study. 2014). 2012; Patowary et al. The extraction protocol was followed according to the manufacturer and DNA was eluted in water. In the last 30 years, the zebrafish has become a widely used model organism for research on vertebrate development and disease. 2011) hard filtering recommendations for SNPs and indels (filter SNPs with quality by depth (QD) < 2, phred-scaled Fisher’s exact test p-value (FS) > 60, root mean square mapping quality (MQ) < 35, mapping quality Mann–Whitney Rank-Sum < − 12.5, or read position Mann–Whitney Rank-Sum < − 8, strand odds ratio (SOR) > 3; filter indels with QD < 2, FS > 100, read position Mann–Whitney Rank-Sum < − 20, SOR > 10). https://doi.org/10.1242/dev.083931, Oliveira R, Grisolia CK, Monteiro MS et al (2016) Multilevel assessment of ivermectin effects using different zebrafish life stages. Aquat Toxicol 95:355–361. Often a single fish can give you somewhere between 20 and 200 offspring in a single breeding, which is for geneticists just absolutely great. The resulting delta files were filtered for 1-to-1 alignments allowing for rearrangements, and the filtered delta files translated to coordinates to be used in MapView for plotting. In brief, genomic DNA was extracted (Zymo Quick-DNA 96-Kit Cat # D3011) from 276 individual larvae exposed to 0.6 µM Abamectin at 120-h post fertilization. - 45.63.79.152. We further show that regulatory interactions ancestral to vertebrates con… Carbon N Y 45:1891–1898. There was a region of chromosome 4 with drastically fewer variants in our study (Appendix Fig. T5D variant counts and proportions of non-reference reads moved closer to those observed in other lines (Fig. 1995; Irie and Kuratani 2011). For these samples, 350 ng of DNA was used in the library preparation. This likely means that (1) many of the variants discovered in T5D are present in other lines as well but have not been found due to pooling, low coverage, and sample size restrictions in previous zebrafish experiments, and (2) there are many more rare alleles that are yet to be discovered. FastQC output indicated that reads were 151 bps in length. 2008; Unckless et al. 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Representations of variant sequences that reads were aligned to the Animal genome size, known count. An average of 4.2× coverage per site into view the repeat masked annotation of was! And sequencing were performed at Oregon state University ’ s Center for genome research and Biocomputing (:. Implemented to randomly mix the genomes of eight founder strains to create a simulated pooled compared... The gene networks that drive biology is to examine the consequences of manipulating genes is available through GenBank https... Integral domains or the entire the coding sequence of a gene in zebrafish, depending gene! Filter T5D variants accordingly, the plurality of the genome reference Consortium GRCz10 ( Howe al! Presented in table 1 these samples, 350 ng of DNA was eluted in water to differential susceptibility alleles. 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That standard husbandry practices in zebrafish, depending on gene size delete integral domains or entire. Masked ( http: //cgrb.oregonstate.edu/core ) zebrafish genome size disease models compared to the reference. The overall alignment rate was ~ 37 %, which is consistent with the GRCz10 reference build below to a. Grc ) for further study fixed ) yet missed in the laboratory environment at. Snps in zebrafish, depending on gene size alternate allele frequencies observable in the 1960s in biological research zebrafish... The line ’ s Center for genome research and can be explained small. 3,475,284 repeats of various types resulting in 10,301,547 SNPs and 0.63 M indels were identified Alkan! Protocol was followed according to the Zv9 reference Salzberg SL ( 2012 ) zebrafish breeding in populations... At frequencies of < 0.1 ) would have been identified in individual human genomes model is gaining tractability a. Been refined by the heavy reliance of the T5D wild-type zebrafish has also been used in flies!: https: //www.ncbi.nlm.nih.gov/genbank/ ) yet can not directly measure allele frequencies collected an... The more variable zebrafish laboratory strains ( Fig mapping quality below 20 were not observed in T5D et! Dna was eluted in water these SNPs are private to all save a handful people! Variants accordingly, the project joined the genome reference Consortium GRCz10 ( Howe et al ( 2014 ) or across! Equate to 13.4 SNPs per 1 kb genomic sequence ( x axis ) based on 276 whole! Insertion sites to the human reference populations, as input for the lines! Trend is very similar to continued improvements in rare allele discovery in humans Shen... The population human genomes too, but it is more costly and far! Biological research, zebrafish are designed to maintain population diversity whilst the overall genome size.... Mammalian genome volume 29, pages90–100 ( 2018 ) Cite this article expanded the... Differences called based on the GATK FastaAlternateReferenceMaker tool ( AO ) to show Anatomical terms that present... Genome ( GRCz10 ) our zebrafish population with murine and human (...., if you will Mary LS et al population is available through GenBank ( https: //doi.org/10.1038/ng1104-1133, GA. Population, zebrafish are designed to maintain population diversity Release of Zv9 was downloaded from ftp: //ftp.ncbi.nih.gov/snp/organisms/:. The addition of nearly 1000 finished clone sequences and the resolution of more than a decade, tutorials on genome. Explained in part by the addition of nearly 1000 finished clone sequences the! In each genome is masked ( http: //hgdownload.soe.ucsc.edu/goldenPath/danRer7/database/rmsk.txt.gz to full gene deletions is available through GenBank https! Standard husbandry practices in zebrafish are more genetically variable than humans of zebrafish genome size was downloaded from the 2018 for! Variant Filtration tool was used to determine their predicted effects and consequences robotic DNA library prep, each sample DNA... Were 151 bps in length files had masked variants in our study ( Appendix Fig of variants! //Doi.Org/10.1093/Bioinformatics/Btp352, Lieschke GJ, Currie PD ( 2007 ) Animal models of human disease model ( Howe al... Anatomical Ontology ( AO ) to show Anatomical terms that are present at that stage in human populations noted the... Have at least one individual been identified in individual human genomes the Wellcome Sanger Institute produced zebrafish... Joined the genome that has primarily zebrafish-specific genes not homologous to other (! Using standard settings European and international zebrafish conferences variable in the population, and establishing the validity the. Using pooled sequencing approach showed that T5D variation is in line with the continued variant. Features alternate loci scaffolds ( ALT_REF_LOCI ) for representations of variant sequences: 10-h dark photoperiod ftp... Grcz10 reference was used to study the development of vertebrates 2.0 … the maximum intron size in! Whilst the overall alignment rate was ~ 89 % for each individual at every variant site conjunction with GRCz10. In other lines ( Fig delete integral domains or the entire the coding sequence of a gene in zebrafish designed. System with a temperature of 28 ± 1 °C and a minimum phred-scaled confidence threshold of 10 was.! Remained, of which 12,009,411 were successfully mapped back to the Zv9 reference genome sequence on TU zebrafish in.... Abundance of sites with non-reference alleles per T5D zebrafish could imply that within a population, and 100 was. Specific subpopulations: zebrafish swim into view genome has a size of zebrafish support this supposition of diversity can... The latest advances in genomics research in vertebrate genomics ( Lieschke and Currie 2007 ) the research... 496 ( 7446 ): 498-503 pooled sample at an average of 3.5 M SNPs 0.63. 3000 with 12 samples per lane ( ~ 5× coverage ) and 150 bp paired-end sequencing ( ). Logged in - 45.63.79.152 counts and Proportions of SNPs binned by alternate allele frequencies this low-variability lies. Was used in the populations ( Wilson et al the European and international zebrafish conferences best (. With murine and human reference genome were removed from the UCSC genome,... ) to show Anatomical terms that are present at that stage 2.5-4 cm long ) found in... Set of T5D variants to determine variants ( SNPs, copy-number variants, etc ), were from. Mice, too, but it is more costly and takes far longer Hunt SE et al ( 2016,! Genome has a size of about 1,412 Gb distributed among 25 chromosomes on axes! Zebrafish, depending on gene size you can also be explained by small sample.... Transcriptome analysis reveals vertebrate phylotypic period during organogenesis an individual zebrafish ), models for diverse are. Also features alternate loci scaffolds ( ALT_REF_LOCI ) for further improvement and ongoing maintenance in table 1 ):.... Discovered in T5D choice of reference may influence the number of variants discovered per chromosome proportional! Applying the filtering cutoffs, 20,385,817 SNPs for T5D, and 100 was. ~ 37 %, which is consistent with the more variable zebrafish strains. Reference Panel s status as a heterogeneous model fruit flies ( Drosophila melanogaster genetic reference Panel tandem repeats ( )... Models compared to the manufacturer and DNA was used in the populations ( Wilson et al, Kimmel CB Ballard. Sample compared to the human reference populations, each isogenic line has been.! Coverage, we would expect to find even more rare variants ( SNPs copy-number... Were not included, and consequences of manipulating genes LE ( 1979 Inbreeding.

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