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July 7, 2019

Extensive mobilome-driven genome diversification in mouse gut-associated Bacteroides vulgatus mpk.

Like many other Bacteroides species, Bacteroides vulgatus strain mpk, a mouse fecal isolate which was shown to promote intestinal homeostasis, utilizes a variety of mobile elements for genome evolution. Based on sequences collected by Pacific Biosciences SMRT sequencing technology, we discuss the challenges of assembling and studying a bacterial genome of high plasticity. Additionally, we conducted comparative genomics comparing this commensal strain with the B. vulgatus type strain ATCC 8482 as well as multiple other Bacteroides and Parabacteroides strains to reveal the most important differences and identify the unique features of B. vulgatus mpk. The genome of B. vulgatus mpk harbors a large and diverse set of mobile element proteins compared with other sequenced Bacteroides strains. We found evidence of a number of different horizontal gene transfer events and a genome landscape that has been extensively altered by different mobilization events. A CRISPR/Cas system could be identified that provides a possible mechanism for preventing the integration of invading external DNA. We propose that the high genome plasticity and the introduced genome instabilities of B. vulgatus mpk arising from the various mobilization events might play an important role not only in its adaptation to the challenging intestinal environment in general, but also in its ability to interact with the gut microbiota.

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July 7, 2019

Exploring structural variants in environmentally sensitive gene families.

Environmentally sensitive plant gene families like NBS-LRRs, receptor kinases, defensins and others, are known to be highly variable. However, most existing strategies for discovering and describing structural variation in complex gene families provide incomplete and imperfect results. The move to de novo genome assemblies for multiple accessions or individuals within a species is enabling more comprehensive and accurate insights about gene family variation. Earlier array-based genome hybridization and sequence-based read mapping methods were limited by their reliance on a reference genome and by misplacement of paralogous sequences. Variant discovery based on de novo genome assemblies overcome the problems arising from a reference genome and reduce sequence misplacement. As de novo genome sequencing moves to the use of longer reads, artifacts will be minimized, intact tandem gene clusters will be constructed accurately, and insights into rapid evolution will become feasible. Copyright © 2016 Elsevier Ltd. All rights reserved.

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July 7, 2019

The Atlantic salmon genome provides insights into rediploidization.

The whole-genome duplication 80 million years ago of the common ancestor of salmonids (salmonid-specific fourth vertebrate whole-genome duplication, Ss4R) provides unique opportunities to learn about the evolutionary fate of a duplicated vertebrate genome in 70 extant lineages. Here we present a high-quality genome assembly for Atlantic salmon (Salmo salar), and show that large genomic reorganizations, coinciding with bursts of transposon-mediated repeat expansions, were crucial for the post-Ss4R rediploidization process. Comparisons of duplicate gene expression patterns across a wide range of tissues with orthologous genes from a pre-Ss4R outgroup unexpectedly demonstrate far more instances of neofunctionalization than subfunctionalization. Surprisingly, we find that genes that were retained as duplicates after the teleost-specific whole-genome duplication 320 million years ago were not more likely to be retained after the Ss4R, and that the duplicate retention was not influenced to a great extent by the nature of the predicted protein interactions of the gene products. Finally, we demonstrate that the Atlantic salmon assembly can serve as a reference sequence for the study of other salmonids for a range of purposes.

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July 7, 2019

First complete genome sequence of the Dutch veterinary Coxiella burnetii strain NL3262, originating from the largest global Q fever outbreak, and draft genome sequence of its epidemiologically linked chronic human isolate NLhu3345937

The largest global Q fever outbreak occurred in The Netherlands during 2007 to 2010. Goats and sheep were identified as the major sources of disease. Here, we report the first complete genome sequence of Coxiella burnetiigoat outbreak strain NL3262 and that of an epidemiologically linked chronic human strain, both having the outbreak-related CbNL01multilocus variable-number tandem-repeat analysis (MLVA) genotype. Copyright © 2016 Kuley et al.

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July 7, 2019

Mechanisms involved in acquisition of blaNDM genes by IncA/C2 and IncFIIY plasmids.

blaNDM genes confer carbapenem resistance and have been identified on transferable plasmids belonging to different incompatibility (Inc) groups. Here we present the complete sequences of four plasmids carrying a blaNDM gene, pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1 and pEC4-NDM-6, from four clinical samples originating from four different patients. Different plasmids carry segments that align to different parts of the blaNDM region found on Acinetobacter plasmids. pKP1-NDM-1 and pEC2-NDM-3, from Klebsiella pneumoniae and Escherichia coli, respectively, were identified as type 1 IncA/C2 plasmids with almost identical backbones. Different regions carrying blaNDM are inserted in different locations in the antibiotic resistance island known as ARI-A and ISCR1 may have been involved in acquisition of blaNDM-3 by pEC2-NDM-3. pECL3-NDM-1 and pEC4-NDM-6, from Enterobacter cloacae and E. coli, respectively, have similar IncFIIY backbones but different regions carrying blaNDM are found in different locations. Tn3-derived Inverted-repeat Transposable Elements (TIME) appear to have been involved in acquisition of blaNDM-6 by pEC4-NDM-6 and the rmtC 16S rRNA methylase gene by IncFIIY plasmids. Characterisation of these plasmids further demonstrates that even very closely related plasmids may have acquired blaNDM genes by different mechanisms. These findings also illustrate the complex relationships between antimicrobial resistance genes, transposable elements and plasmids and provide insights into the possible routes for transmission of blaNDM genes amongst species of the Enterobacteriaceae family. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Complete nucleotide sequence of pH11, an IncHI2 plasmid conferring multi-antibiotic resistance and multi-heavy metal resistance genes in a clinical Klebsiella pneumoniae isolate.

The complete 284,628bp sequence of pH11, an IncHI2 plasmid, was determined through single-molecule, real-time (SMRT) sequencing. Harbored by a clinical Klebsiella pneumoniae strain H11, and isolated in Beijing, this plasmid contains multiple antibiotic resistance genes, including catA2, aac(6′)-Ib, strB, strA, dfrA19, blaTEM-1, blaSHV-12, sul1, qacE delta 1, ereA, arr2, and aac3. The aac(6′)-Ib is carried by a class I integron. Plasmid pH11 also carries several genes associated with resistance to heavy metals, such as tellurium, mercury, cobalt, zinc, nickel, copper, lead and cadmium. This plasmid exhibits numerous characteristics, including HipBA and RelBE toxin-antitoxin systems, two major transfer (Tra) regions closely related to those of Salmonella enterica serovar plasmid pRH-R27, a type II restriction modification system (EcoRII R-M system), several methyltransferases and methylases and genes encoding Hha and StpA. These characteristics suggest that pH11 may adapt to various hosts and environments. Multiple insertion sequence elements, transposases, recombinases, resolvases and integrases are scattered throughout pH11. The presence of these genes may indicate that horizontal gene transfer occurs frequently in pH11 and thus may facilitate the dissemination of antimicrobial resistance determinants. Our data suggest that pH11 is a chimera gradually assembled through the integration of different horizontally acquired DNA segments via transposition or homologous recombination. Copyright © 2016 Elsevier Inc. All rights reserved.

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July 7, 2019

An improved genome assembly of Azadirachta indica A. Juss.

Neem (Azadirachta indica A. Juss.), an evergreen tree of the Meliaceae family, is known for its medicinal, cosmetic, pesticidal and insecticidal properties. We had previously sequenced and published the draft genome of the plant, using mainly short read sequencing data. In this report, we present an improved genome assembly generated using additional short reads from Illumina and long reads from Pacific Biosciences SMRT sequencer. We assembled short reads and error corrected long reads using Platanus, an assembler designed to perform well for heterozygous genomes. The updated genome assembly (v2.0) yielded 3- and 3.5-fold increase in N50 and N75, respectively; 2.6-fold decrease in the total number of scaffolds; 1.25-fold increase in the number of valid transcriptome alignments; 13.4-fold less mis-assembly and 1.85-fold increase in the percentage repeat, over the earlier assembly (v1.0). The current assembly also maps better to the genes known to be involved in the terpenoid biosynthesis pathway. Together, the data represents an improved assembly of the A. indica genome. The raw data described in this manuscript are submitted to the NCBI Short Read Archive under the accession numbers SRX1074131, SRX1074132, SRX1074133, and SRX1074134 (SRP013453). Copyright © 2016 Author et al.

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July 7, 2019

Diverse CRISPR-Cas responses and dramatic cellular DNA changes and cell death in pKEF9-conjugated Sulfolobus species.

The Sulfolobales host a unique family of crenarchaeal conjugative plasmids some of which undergo complex rearrangements intracellularly. Here we examined the conjugation cycle of pKEF9 in the recipient strain Sulfolobus islandicus REY15A. The plasmid conjugated and replicated rapidly generating high average copy numbers which led to strong growth retardation that was coincident with activation of CRISPR-Cas adaptation. Simultaneously, intracellular DNA was extensively degraded and this also occurred in a conjugated ?cas6 mutant lacking a CRISPR-Cas immune response. Furthermore, the integrated forms of pKEF9 in the donor Sulfolobus solfataricus P1 and recipient host were specifically corrupted by transposable orfB elements, indicative of a dual mechanism for inactivating free and integrated forms of the plasmid. In addition, the CRISPR locus of pKEF9 was progressively deleted when conjugated into the recipient strain. Factors influencing activation of CRISPR-Cas adaptation in the recipient strain are considered, including the first evidence for a possible priming effect in Sulfolobus. The 3-Mbp genome sequence of the donor P1 strain is presented..© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019

Dynamics of mutations during development of resistance by Pseudomonas aeruginosa against five antibiotics.

Pseudomonas aeruginosa is an opportunistic pathogen that causes considerable morbidity and mortality, specifically in the intensive care. Antibiotic resistant variants of this organism are more difficult to treat and cause substantial extra costs compared to susceptible strains. In the laboratory, P. aeruginosa rapidly developed resistance against five medically relevant antibiotics upon exposure to step-wise increasing concentrations. At several time points during the acquisition of resistance samples were taken for whole genome sequencing. The increase of MIC for ciprofloxacin was linked to specific mutations in gyrA, parC and gyrB, appearing sequentially. In the case of tobramycin, mutations were induced in fusA, HP02880, rplB and capD The MIC for the beta-lactam compounds meropenem, ceftazidime and the combination piperacillin/tazobactam correlated linearly with the beta-lactamase activity, but not always with individual mutations. The genes that were mutated during development of beta-lactam resistance differed for each antibiotic. A quantitative relationship between the frequency of mutations and the increase in resistance could not be established for any of the antibiotics. When the adapted strains are grown in the absence of the antibiotic, some mutations remained and others were reverted, but this reversal did not necessarily lower the MIC. The increased MIC came at the cost of moderately reduced cellular functions, or somewhat lower growth rate. In all cases except ciprofloxacin, the increase of resistance seems to be the result of a complex interaction between several cellular systems, rather than individual mutations. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Elucidating the triplicated ancestral genome structure of radish based on chromosome-level comparison with the Brassica genomes.

This study presents a chromosome-scale draft genome sequence of radish that is assembled into nine chromosomal pseudomolecules. A comprehensive comparative genome analysis with the Brassica genomes provides genomic evidences on the evolution of the mesohexaploid radish genome. Radish (Raphanus sativus L.) is an agronomically important root vegetable crop and its origin and phylogenetic position in the tribe Brassiceae is controversial. Here we present a comprehensive analysis of the radish genome based on the chromosome sequences of R. sativus cv. WK10039. The radish genome was sequenced and assembled into 426.2 Mb spanning >98 % of the gene space, of which 344.0 Mb were integrated into nine chromosome pseudomolecules. Approximately 36 % of the genome was repetitive sequences and 46,514 protein-coding genes were predicted and annotated. Comparative mapping of the tPCK-like ancestral genome revealed that the radish genome has intermediate characteristics between the Brassica A/C and B genomes in the triplicated segments, suggesting an internal origin from the genus Brassica. The evolutionary characteristics shared between radish and other Brassica species provided genomic evidences that the current form of nine chromosomes in radish was rearranged from the chromosomes of hexaploid progenitor. Overall, this study provides a chromosome-scale draft genome sequence of radish as well as novel insight into evolution of the mesohexaploid genomes in the tribe Brassiceae.

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July 7, 2019

Next-generation sequencing-based detection of germline L1-mediated transductions.

While active LINE-1 (L1) elements possess the ability to mobilize flanking sequences to different genomic loci through a process termed transduction influencing genomic content and structure, an approach for detecting polymorphic germline non-reference transductions in massively-parallel sequencing data has been lacking.Here we present the computational approach TIGER (Transduction Inference in GERmline genomes), enabling the discovery of non-reference L1-mediated transductions by combining L1 discovery with detection of unique insertion sequences and detailed characterization of insertion sites. We employed TIGER to characterize polymorphic transductions in fifteen genomes from non-human primate species (chimpanzee, orangutan and rhesus macaque), as well as in a human genome. We achieved high accuracy as confirmed by PCR and two single molecule DNA sequencing techniques, and uncovered differences in relative rates of transduction between primate species.By enabling detection of polymorphic transductions, TIGER makes this form of relevant structural variation amenable for population and personal genome analysis.

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July 7, 2019

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida.

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n?=?14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

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July 7, 2019

Population scale mapping of transposable element diversity reveals links to gene regulation and epigenomic variation.

Variation in the presence or absence of transposable elements (TEs) is a major source of genetic variation between individuals. Here, we identified 23,095 TE presence/absence variants between 216 Arabidopsis accessions. Most TE variants were rare, and we find these rare variants associated with local extremes of gene expression and DNA methylation levels within the population. Of the common alleles identified, two thirds were not in linkage disequilibrium with nearby SNPs, implicating these variants as a source of novel genetic diversity. Many common TE variants were associated with significantly altered expression of nearby genes, and a major fraction of inter-accession DNA methylation differences were associated with nearby TE insertions. Overall, this demonstrates that TE variants are a rich source of genetic diversity that likely plays an important role in facilitating epigenomic and transcriptional differences between individuals, and indicates a strong genetic basis for epigenetic variation.

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July 7, 2019

Microevolution of monophasic Salmonella Typhimurium during epidemic, United Kingdom, 2005-2010.

Microevolution associated with emergence and expansion of new epidemic clones of bacterial pathogens holds the key to epidemiologic success. To determine microevolution associated with monophasic Salmonella Typhimurium during an epidemic, we performed comparative whole-genome sequencing and phylogenomic analysis of isolates from the United Kingdom and Italy during 2005-2012. These isolates formed a single clade distinct from recent monophasic epidemic clones previously described from North America and Spain. The UK monophasic epidemic clones showed a novel genomic island encoding resistance to heavy metals and a composite transposon encoding antimicrobial drug resistance genes not present in other Salmonella Typhimurium isolates, which may have contributed to epidemiologic success. A remarkable amount of genotypic variation accumulated during clonal expansion that occurred during the epidemic, including multiple independent acquisitions of a novel prophage carrying the sopE gene and multiple deletion events affecting the phase II flagellin locus. This high level of microevolution may affect antigenicity, pathogenicity, and transmission.

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July 7, 2019

A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer.

Although human LINE-1 (L1) elements are actively mobilized in many cancers, a role for somatic L1 retrotransposition in tumor initiation has not been conclusively demonstrated. Here, we identify a novel somatic L1 insertion in the APC tumor suppressor gene that provided us with a unique opportunity to determine whether such insertions can actually initiate colorectal cancer (CRC), and if so, how this might occur. Our data support a model whereby a hot L1 source element on Chromosome 17 of the patient’s genome evaded somatic repression in normal colon tissues and thereby initiated CRC by mutating the APC gene. This insertion worked together with a point mutation in the second APC allele to initiate tumorigenesis through the classic two-hit CRC pathway. We also show that L1 source profiles vary considerably depending on the ancestry of an individual, and that population-specific hot L1 elements represent a novel form of cancer risk. © 2016 Scott et al.; Published by Cold Spring Harbor Laboratory Press.

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