Whole genome sequence

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Draft Whole-Genome Sequence of the Anthracene-Degrading Strain Mycolicibacterium frederiksbergense LB501T, Isolated from a Polycyclic Aromatic Hydrocarbon-Contaminated Soil

Draft Whole-Genome Sequence of the Anthracene-Degrading Strain Mycolicibacterium frederiksbergense LB501T, Isolated from a Polycyclic Aromatic Hydrocarbon-Contaminated Soil

SSPACE-Longread version 1.1. The other three scaffolds correspond to plasmids, one 341,437-bp plasmid (G 1C content, 65.8%), one 268,343-bp plasmid (G1C content, 65.2%), and one 16,966-bp plasmid (G 1C content, 66.1%). The chromosome and the 341,437-bp plasmid each contain one within-scaffold gap of 2,142 bp (3,206,107 to 3,208,248) and 491 bp (862 to 1,352), respectively. The whole-genome sequence encom- passes 6,440 coding sequences (CDS), 2 rRNA operons (16S, 23S, and 5S rRNAs), 2 non- coding RNAs, and 46 tRNA genes. Two chromosomally encoded catabolic regions of approximately 99 and 33 kb were identi fied at positions 737,825 to 836,329 and 3,866,266 to 3,899,381, respectively, which contain several gene clusters required for complete PAH biodegradation, such as phenanthrene, anthracene, and pyrene. In particular, the 99-kb catabolic region presents a gene structure and organization similar to those of the 150-kb catabolic region A from Mycobacterium vanbaalenii PYR-1 (10, 11). These regions contain several ring-hydroxylating dioxygenases (RHDs), including nidAB, nidA3B3, and pdoA2B2, responsible for initial dioxygenation in the ring cleavage process of low- and high-molecular-weight PAHs (12, 13). The pht cluster (phtRAaAbBAcAd) (14) and the b-ketoadipate pathway cluster (pcaJICDBGHR) (15, 16) were identi fied in the major catabolic region, which demonstrates that anthracene degradation by strain LB501T proceeds through the o-phthalate degradation path- way (phthalate 3,4-dioxygenase) and ortho-cleavage of protocatechuate (protocate- chuic acid [PCA] 3,4-dioxygenase), as previously suggested (17). Moreover, the ge- nome sequence harbors multiple genes involved in heavy metal resistance, such as copper (copA, copC, and copD), arsenic (arsRBCDA), and mercury (merA and merB). The presence of these metal resistance genes suggests that Mycolicibacterium freder- iksbergense strain LB501T could be a promising microorganism for bioaugmentation of soils contaminated with both PAHs and heavy metals.
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Whole-genome sequence of Serratia symbiotica strain CWBI-2.3T, a free-living symbiont of the Black Bean Aphid Aphis fabae

Whole-genome sequence of Serratia symbiotica strain CWBI-2.3T, a free-living symbiont of the Black Bean Aphid Aphis fabae

Received 9 July 2014 Accepted 30 July 2014 Published 21 August 2014 Citation Foray V, Grigorescu AS, Sabri A, Haubruge E, Lognay G, Francis F, Fauconnier M-L, Hance T, Thonart P. 2014. Whole-genome sequence of Serratia symbiotica strain CWBI-2.3 T , a free-living symbiont of the black bean aphid Aphis fabae. Genome Announc. 2(4):e00767-14. doi:10.1128/genomeA.00767-14. Copyright © 2014 Foray et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license . Address correspondence to Vincent Foray, vincent.foray@uclouvain.be.
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Whole-genome sequence-based analysis of thyroid function

Whole-genome sequence-based analysis of thyroid function

Erratum: Whole-genome sequence-based analysis of thyroid function Peter N. Taylor, Eleonora Porcu, Shelby Chew, Purdey J. Campbell, Michela Traglia, Suzanne J. Brown, Benjamin H. Mullin, Hashem A. Shihab, Josine Min, Klaudia Walter, Yasin Memari, Jie Huang, Michael R. Barnes, John P. Beilby, Pimphen Charoen, Petr Danecek, Frank Dudbridge, Vincenzo Forgetta, Celia Greenwood, Elin Grundberg, Andrew D. Johnson, Jennie Hui, Ee M. Lim, Shane McCarthy, Dawn Muddyman, Vijay Panicker, John R.B. Perry, Jordana T. Bell, Wei Yuan, Caroline Relton, Tom Gaunt, David Schlessinger, Goncalo Abecasis, Francesco Cucca, Gabriela L. Surdulescu, Wolfram Woltersdorf, Eleftheria Zeggini, Hou-Feng Zheng,
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Whole-Genome Sequence of a Brucella pinnipedialis Sequence Type 54 Strain Isolated from a Hooded Seal ( Cystophora cristata ) from the North Atlantic Ocean, Norway

Whole-Genome Sequence of a Brucella pinnipedialis Sequence Type 54 Strain Isolated from a Hooded Seal ( Cystophora cristata ) from the North Atlantic Ocean, Norway

Whole-Genome Sequence of a Brucella pinnipedialis Sequence Type 54 Strain Isolated from a Hooded Seal (Cystophora cristata) from the North Atlantic Ocean, Norway Michel S. Zygmunt , a Gilles Vergnaud , b Axel Cloeckaert a a INRAE, UMR ISP, Université de Tours, Nouzilly, France

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Draft whole-genome sequence of the anthracene-degrading strain Mycolicibacterium frederiksbergense LB501T, isolated from a polycyclic aromatic hydrocarbon-contaminated soil

Draft whole-genome sequence of the anthracene-degrading strain Mycolicibacterium frederiksbergense LB501T, isolated from a polycyclic aromatic hydrocarbon-contaminated soil

SSPACE-Longread version 1.1. The other three scaffolds correspond to plasmids, one 341,437-bp plasmid (G 1C content, 65.8%), one 268,343-bp plasmid (G1C content, 65.2%), and one 16,966-bp plasmid (G 1C content, 66.1%). The chromosome and the 341,437-bp plasmid each contain one within-scaffold gap of 2,142 bp (3,206,107 to 3,208,248) and 491 bp (862 to 1,352), respectively. The whole-genome sequence encom- passes 6,440 coding sequences (CDS), 2 rRNA operons (16S, 23S, and 5S rRNAs), 2 non- coding RNAs, and 46 tRNA genes. Two chromosomally encoded catabolic regions of approximately 99 and 33 kb were identi fied at positions 737,825 to 836,329 and 3,866,266 to 3,899,381, respectively, which contain several gene clusters required for complete PAH biodegradation, such as phenanthrene, anthracene, and pyrene. In particular, the 99-kb catabolic region presents a gene structure and organization similar to those of the 150-kb catabolic region A from Mycobacterium vanbaalenii PYR-1 (10, 11). These regions contain several ring-hydroxylating dioxygenases (RHDs), including nidAB, nidA3B3, and pdoA2B2, responsible for initial dioxygenation in the ring cleavage process of low- and high-molecular-weight PAHs (12, 13). The pht cluster (phtRAaAbBAcAd) (14) and the b-ketoadipate pathway cluster (pcaJICDBGHR) (15, 16) were identi fied in the major catabolic region, which demonstrates that anthracene degradation by strain LB501T proceeds through the o-phthalate degradation path- way (phthalate 3,4-dioxygenase) and ortho-cleavage of protocatechuate (protocate- chuic acid [PCA] 3,4-dioxygenase), as previously suggested (17). Moreover, the ge- nome sequence harbors multiple genes involved in heavy metal resistance, such as copper (copA, copC, and copD), arsenic (arsRBCDA), and mercury (merA and merB). The presence of these metal resistance genes suggests that Mycolicibacterium freder- iksbergense strain LB501T could be a promising microorganism for bioaugmentation of soils contaminated with both PAHs and heavy metals.
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Whole-genome sequence of a colombian Acinetobacter baumannii strain, a coproducer of OXA-72 and OXA-255-like carbapenemases

Whole-genome sequence of a colombian Acinetobacter baumannii strain, a coproducer of OXA-72 and OXA-255-like carbapenemases

23. Werneck JS, Picão RC, Carvalhaes CG, Cardoso JP, Gales AC. 2011. OXA-72-producing Acinetobacter baumannii in Brazil: a case report. J Antimicrob Chemother 66:452– 454. https://doi.org/10.1093/jac/dkq462 . 24. Dortet L, Bonnin RA, Girlich D, Imanci D, Bernabeu S, Fortineau N, Naas T. 2015. Whole-genome sequence of a European Clone II and OXA-72- producing Acinetobacter baumannii strain from Serbia. Genome An- nounc 3(6):e01390-15. https://doi.org/10.1128/genomeA.01390-15 . 25. Georgescu M, Gheorghe I, Dudu A, Czobor I, Costache M, Cristea VC,

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A whole-genome sequence and transcriptome perspective on HER2-positive breast cancers

A whole-genome sequence and transcriptome perspective on HER2-positive breast cancers

Mutations in cis-regulatory regions . Annotation of somatic variants that are located in cis-regulatory regions was performed using OncoCis 32 , based on human mammary epithelial cell epigenomic data sets. Annotated somatic variants were further processed to classify those in potential promoter or enhancer regions. Somatic SNVs in potential promoter regions were extracted if they localize around H3K4me3 histone marks. Somatic variants in potential enhancer regions were extracted if they localize around H3K4me1 or H3K27ac histone marks. In addition, mutations were further annotated using FANTOM5 predictions 61 , which uses cap analysis of gene expression to detect potentially active transcription from promotor or enhancer regions. In both cases (promotors and enhancers regions) we computed, per patient, the fraction of somatic SNVs located in these regions (that is, number of SNVs in regions divided by the total number of SNVs in patient). Copy-number analysis . The analysis of CNVs from whole-genome sequence data was performed in three steps. First, after reads alignment using the Burrows- Wheeler alignment tool (BWA), raw read counts at each genomic position were corrected by GC content using the approach described by Benjamini and Speed 62 . We slightly improved the construction of the empirical dependency model by using only raw counts in mappable 63 regions, by sampling positions (10 7 ) over the whole
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Whole genome sequence of mycoplasma mycoides subsp. Mycoides LC : application to the development of new diagnostic tools and heterologous gene expression

Whole genome sequence of mycoplasma mycoides subsp. Mycoides LC : application to the development of new diagnostic tools and heterologous gene expression

1.1.2 Genome characteristics and phylogeny The Mycoplasmas have evolved from more conventional progenitors in the Firmicutes taxon by a process of massive genome reduction (Glass et al., 2006); their genome size ranges from 0.58-2.2 Mbp. They have been shown to be closely related to gram positive bacteria with a low G + C content in their genome. The reassignment, in most mollicutes of UGA from a stop codon to a tryptophan codon, a feature found in mitochondria, is the apparent outcome of codon reassignment under strong A+T pressure. Not all mollicutes do share this property; the phylogenetically early Acholeplasma and Phytoplasma use the conventional UGG codon for tryptophan retaining UGA as a stop codon (Razin et al., 1998). Mycoplasma genomes have a limited coding capacity, and as a consequence, they lack many enzymatic pathways characteristic of many bacteria. For example, they lack the pathways for the biosynthesis of purines, a functional tricarboxylic acid cycle and a cytochrome mediated electron transport system (Dybvig and Voelker, 1996; Maniloff, 1996). Mycoplasmas genome size illustrates extreme biological gene economy, imposing complex nutritional requirements for replications and survival. This implies a dependence on external supplies for biosynthetic precursors including amino acids, nucleotides, fatty acids and sterols. The nature of the selective pressure for repeated genome reduction during mycoplasma phylogeny is not known. These features make mycoplasmas suitable research objects in the determination of the minimal gene set required for independent life and to be used as a model in the creation of artificial cells devoid of all non-essential genes (Glass et al., 2006).
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Colon cancer-derived oncogenic EGFR G724S mutant identified by whole genome sequence analysis is dependent on asymmetric dimerization and sensitive to cetuximab

Colon cancer-derived oncogenic EGFR G724S mutant identified by whole genome sequence analysis is dependent on asymmetric dimerization and sensitive to cetuximab

Findings Activation of the epidermal growth factor receptor (EGFR) oncoprotein, a member of the ErbB family of re- ceptor tyrosine kinases, is among the most common oncogenic driving events in human cancer [1]. Genomic mechanisms for activating the EGFR gene include nu- cleotide substitutions and in-frame insertions/deletions of the kinase domain in lung adenocarcinoma and papil- lary thyroid carcinomas, and multi-exonic deletions (exons 2 through 7: EGFR variant III or vIII), nucleotide substitutions of the extracellular domain and carboxyl terminal deletions in glioblastoma [2-6]. EGFR is also ac- tivated by high-copy amplifications in many epithelial cancer types, prominently in lung and upper gastrointes- tinal carcinomas as well as glioblastoma and head and neck cancer [7-10]. Furthermore, EGFR protein is over- expressed in many cancers even without evidence of fo- cused genomic alteration, as observed in many cases of colorectal carcinoma where EGFR kinase domain muta- tions were found in only 3 out of 224 cases, 1.3% sub- jected to whole exome sequencing [11,12]. Given the elevated expression and genomic alterations present in EGFR, multiple cancer therapies have targeted EGFR, as both its kinase activity and its dependence on extracellu- lar ligand signaling have rendered EGFR vulnerable to therapeutic intervention. FDA-approved EGFR targeted inhibitors include the low-molecular-weight ATP- competitive kinase inhibitors, such as gefitinib and erlo- tinib, and humanized monoclonal antibodies directed against the extracellular domain, notably cetuximab and panitumumab [13]. Although high-level expression of EGFR ligands and/or increased EGFR gene copy num- bers may be predictive markers for antitumor response by cetuximab in colon cancer [14-16], and patients with RAS driven cancers are known not to benefit from cetuximab treatment, a clear molecular explanation of cancer response to cetuximab has remained elusive. Genomic studies identify G724S mutant in colorectal carcinomas
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First reported outbreak of Duck atadenovirus A tracheobronchitis in 3-week-old ducklings in Québec including whole genome sequence of the virus

First reported outbreak of Duck atadenovirus A tracheobronchitis in 3-week-old ducklings in Québec including whole genome sequence of the virus

Considering that no entire viral genome sequence of DAdV-1 has been previously reported in Canada, we decided to estab- lish whether our virus was similar to what has been previously reported worldwide. Thus, an attempt was made to sequence the entire viral genome directly from the clinical samples using MiSeq Illumina technology (Illumina, Vancouver, British Columbia) to allow us to better characterize our DAdV-1 strain, named FMV19-2234581. Briefly, the tissue was lysed for 10 min using glass beads. Then, the tissue lysate was centri- fuged at low speed to remove large particles. The supernatant was placed on a sucrose cushion and then ultracentrifuged in an AirFuge microultracentrifuge (Beckman Coulter, Mississauga, Ontario) for 1 h to concentrate the virus. The pelleted virus was resuspended in phosphate-buffered saline (PBS) and the DNA was extracted using the Zymo RNA/DNA extraction kit (Cedarlane, Burlington, Ontario). The DNA was quanti- fied using a Qubit fluorometer with the HS DNA kit, then a library was generated with a Nextera XT kit (Illumina). The library was sequenced using a MiSeq apparatus with a 600 v3 cartridge. The complete genome was obtained using the de novo metagenomic application plus the extend contigs option of the CLC Genomic Workbench, Version 12.0.3 software (Qiagen, Toronto, Ontario). The annotations were transferred using the reference strain Duck adenovirus 1 isolate D11-JW-032 (KJ452172) in Geneious Prime software, Version 2019.2.1 (Biomatters, Newark, New Jersey, USA). The nucleotide homol- ogy and PHYLM phylogenetic tree were made from a MAFFT alignment.
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Detection of long repeat expansions from PCR-free whole-genome sequence data

Detection of long repeat expansions from PCR-free whole-genome sequence data

repeats from 152 samples with eight other pathogenic repeat ex- pansions. In total, we examined five repeat motifs (CTG, GAA, CGG, CAG, and GGCCCC) at nine different genomic locations and demonstrated that ExpansionHunter can detect repeat expan- sions in a variety of sequence contexts. It is particularly important that our method works on the very high (100%) GC repeats in FMR1 (CGG) and C9orf72 (GGCCCC) genes, where both coverage biases and error rates may be elevated. Comparing our size esti- mates with Southern blot experiments indicated that our method may underestimate sizes of some very long repeats, particularly those in the FMR1 and AR genes (Fig. 4; Supplemental Table 7 ). This discrepancy could be due to mosaicism of expanded FMR1 re- peats (in fact, several of the samples with FMR1 expansions were identified as mosaic in the Coriell database). In addition to mosa- icism, other factors such as higher error rates and GC biases may play a role in causing this method to underestimate the size of these long repeats. Still, the FMR1 expansions were generally sized as being longer compared to the FMR1 premutations, indicating that it may be possible to calibrate the size estimates and account for errors not related to mosaicism. Future work will concentrate on quantifying this behavior and improving the size estimates for these long repeats.
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Whole genome sequence of Vibrio cholerae directly from dried spotted filter paper

Whole genome sequence of Vibrio cholerae directly from dried spotted filter paper

Automated annotation was performed using PROKKA v1.11 [ 19 ] and a genus-specific data- base from RefSeq [ 20 ]. Variation detection was performed using SamtoolsMpileup v0.1.19 [ 21 ] with parameters “-d 1000 -DSugBf” and bcftools v0.1.19 [ 22 ] to produce a BCF file of all variant sites. All bases were filtered to remove those with uncertainty in the base call. The bcftools variant quality score was required to be greater than 50 and the mapping quality to be greater than 30. If all reads did not give the same base call, the allele frequency, as calculated by bcftools, was required to be either 0 for bases called the same as the reference, or 1 for bases called as a single nucleotide polymorphism (SNP) (af1 < 0.95). The majority base call was required to be pres- ent in at least 75% of reads mapping at the base, (ratio < 0.75), and the minimum mapping depth required was 4 reads, at least two of which had to map to each strand (depth < 4, depth_strand< 2). Finally, strand bias was required to be less than 0.001, map bias less than 0.001 and tail bias less than 0.001. If any of these filters were not met, the base was called as uncertain. A pseudo-genome was constructed by substituting the base call at each site (variant and non-variant) in the BCF file into the reference genome and any site called as uncertain was substituted with an N. Insertions with respect to the reference genome were ignored and deletions with respect to the reference genome were filled with N’s in the pseudo-genome to keep it aligned and the same length as the reference genome used for read mapping. Mapping was visualised with Artemis [ 23 ] and ACT [ 24 ].
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Draft Whole-Genome Shotgun Sequence of Streptomyces sp. Strain ETH9427

Draft Whole-Genome Shotgun Sequence of Streptomyces sp. Strain ETH9427

6. Zhou Y, Liang Y, Lynch KH, Dennis JJ, Wishart DS. 2011. PHAST: a fast phage search tool. Nucleic Acids Res 39:W347–W352. https://doi.org/10 .1093/nar/gkr485 . 7. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. 2007. DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81–91.

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Genome-Wide Association Study of HIV Whole Genome Sequences Validated using Drug Resistance

Genome-Wide Association Study of HIV Whole Genome Sequences Validated using Drug Resistance

Overall, our results provide a clear proof of concept on the use of GWAS within HIV and other viruses whole genome sequence data. The smaller genome size, compared to humans, means that substantially smaller samples were needed to identify associated variants. Power is also greater because sequencing allows one to test the association with the causal variant, rather than the proxy SNPs often used in human GWAS to capture several nearby correlated SNPs. With a larger percentage of the genome transcribed there should also be a larger proportion of functionally relevant variants. Additionally, viruses can themselves be used as model organisms and can be genetically modified, allowing for functional validation of identified variants in a way that cannot be performed in humans. However, these benefits of performing GWAS within viruses should not ignore the valuable lessons from human genomics, especially the need to quickly establish large sample sizes through internationally collaborative research (see [ 25 , 26 ]). A focus on setting up standardised quality control pipelines, making GWAS results publically available in the form of SNP summary statistics, and pooling samples into mega- analyses (rather than meta-analysing separate studies) should be the aim of those groups gener- ating HIV and other virus genomes.
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Complete genome sequence of Mycobacterium sp. Strain 3519A

Complete genome sequence of Mycobacterium sp. Strain 3519A

M ycobacterium sp. strain 3519A is a rapidly growing scotochromogenic acid-fast bacillus isolated from the sputum of a Cambodian patient with a pulmonary infection. We sequenced and analyzed the whole-genome sequence of strain 3519A in order to describe its genomic content and to determine its phylogenetic relationships for facilitating its detection and identification.

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Evolution of gene expression after whole-genome duplication: new insights from the spotted gar genome

Evolution of gene expression after whole-genome duplication: new insights from the spotted gar genome

Neuhauss, 2014). With more than 30,000 species, living teleost fish occupy a wide diversity of aquatic habitats, including the most extreme ones, like deep-sea vents, frigid Antarctic waters, acid hot springs, and ephemeral pools. The relation of the TGD and teleost biodiversity, however, remains intricate and is uncoupled in geological time (Santini et al., 2009; Clarke et al., 2016). After genome duplication by autopolyploidy, both duplicated gene copies initially encode proteins with identical sequences and expression patterns; over time, some gene pairs revert to single copy (loss of one of the duplicate copies), while members of other pairs evolve new functions
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ShallowHRD: detection of homologous recombination deficiency from shallow whole genome sequencing

ShallowHRD: detection of homologous recombination deficiency from shallow whole genome sequencing

Genome analysis ShallowHRD: detection of homologous recombination deficiency from shallow whole genome sequencing Alexandre Eeckhoutte 1,2, *, Alexandre Houy 1,2 , Elodie Manie´ 1,2 , Manon Reverdy 1,2 , Ivan Bie`che 3 , Elisabetta Marangoni 2,4 , Oumou Goundiam 2,4 , Anne Vincent-Salomon 5 , Dominique Stoppa-Lyonnet 1,6 , Franc¸ois-Cle´ment Bidard 7,8 , Marc-Henri Stern 1,2,3 and Tatiana Popova 1,2

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Genome sequence of the cluster root forming white lupin;

Genome sequence of the cluster root forming white lupin;

less than 5 genes) corresponding to independent sets of blocks sharing orthologous relationships in modern species. In the third step, conserved gene pairs or conserved groups of gene-to-gene adjacencies defining identical chromosome-to- chromosome relationships between all the extant genomes are merged into Conserved Ancestral Regions (CARs). CARs are then merged into protochromosomes based on partial synteny observed between a subset (not all) of the investigated species. The ancestral karyotype can be considered as a ‘median’ or ‘intermediate’ genome consisting of protochromosomes defining a clean reference gene order common to the existent species investigated. From the reconstructed ancestral karyotype an evolutionary scenario was then inferred taking into account the fewest number of genomic rearrangements (including inversions, deletions, fusions, fissions, translocations), which may have operated between the inferred ancestors and the modern genomes. Additional information are provided in Supplementary Note 4.3.
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The rainbow trout genome provides novel insights into evolution after whole-genome duplication in vertebrates.

The rainbow trout genome provides novel insights into evolution after whole-genome duplication in vertebrates.

Despite this 100 My of evolution since the Ss4R, we found many evidences of an extreme stability of the two ancestral genome copies in the rainbow trout genome. At the chromosome level, no pervasive rearrangements were observed, and the structure of the Ss4R paralogous sequences remains surprisingly well conserved in sequence identity and gene order on chromosomes. Together these observations show that gene fractionation does not involve many genomic rearrangements such as inversions or translocations that would modify the order of genes in the genome, or large deletions that would result in long clusters of singletons. At the gene level we estimated the number of genes in the pre-Ss4R salmonid ancestor to be B31,000 genes. Interestingly, the zebrafish genome, the teleost fish with the most exhaustive and well-supported protein-coding gene annotation to date, contains B26,000 genes. This figure of 31,000 genes estimated here for the ancestral salmonid genome is compatible with the modern estimate in zebrafish, since, being 100 My closer to the event, the pre-Ss4R ancestral salmonid was likely to contain more duplicated gene copies remaining from the teleost Ts3R WGD. After 100 My of evolution since the Ss4R, gene fractionation only affected half of the ancestral Ss4R ohnologues, which have now returned to a single-copy state, resulting in an average gene inactivation rate of B170 genes per
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Construction of a duck whole genome radiation hybrid panel : an aid for NGS whole genome assembly and a contribution to avian comparative maps

Construction of a duck whole genome radiation hybrid panel : an aid for NGS whole genome assembly and a contribution to avian comparative maps

in situ PCR. Complete amplification allows the generation of dense template clusters. The last step of the workflow is the sequencing. A flow cell containing millions of unique clusters is loaded into the sequencer for automated cycles of extension and imaging. The first cycle of sequencing consists in the incorporation of a single fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base, each of the 4 possible bases having its specific fluorescence wavelength. Any signal above the background identifies the physical location of a cluster and the fluorescent emission identifies which of the four bases is incorporated at that position. To initiate the first sequence circle, all four labeled reversible terminator, sequencing primer and DNA polymerase are added into the flow cell. All unincorporated reagents are washed away and the image of emitted fluorescence from each cluster is captured after laser excitation. The blocked 3’ terminus and the fluorophore from each incorporated base are then removed. To initiate the second sequence cycle, all four labeled reversible terminators and DNA polymerase are added again and the process above is repeated until the end of the run. Base calls are derived with an algorithm that identified the color emission over time for each cluster position on the flow cell.
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