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Asset Tag: Hybrid assembly

July 7, 2019

New insights into structural organization and gene duplication in a 1.75-Mb genomic region harboring the a-gliadin gene family in Aegilops tauschii, the source of wheat D genome.

Among the wheat prolamins important for its end-use traits, a-gliadins are the most abundant, and are also a major cause of food-related allergies and intolerances. Previous studies of various wheat species estimated that between 25 and 150 a-gliadin genes reside in the Gli-2 locus regions. To better understand the evolution of this complex gene family, the DNA sequence of a 1.75-Mb genomic region spanning the Gli-2 locus was analyzed in the diploid grass, Aegilops tauschii, the ancestral source of D genome in hexaploid bread wheat. Comparison with orthologous regions from rice, sorghum, and Brachypodium revealed rapid and dynamic changes only occurring to the Ae. tauschii Gli-2 region, including insertions of high numbers of non-syntenic genes and a high rate of tandem gene duplications, the latter of which have given rise to 12 copies of a-gliadin genes clustered within a 550-kb region. Among them, five copies have undergone pseudogenization by various mutation events. Insights into the evolutionary relationship of the duplicated a-gliadin genes were obtained from their genomic organization, transcription patterns, transposable element insertions and phylogenetic analyses. An ancestral glutamate-like receptor (GLR) gene encoding putative amino acid sensor in all four grass species has duplicated only in Ae. tauschii and generated three more copies that are interspersed with the a-gliadin genes. Phylogenetic inference and different gene expression patterns support functional divergence of the Ae. tauschii GLR copies after duplication. Our results suggest that the duplicates of a-gliadin and GLR genes have likely taken different evolutionary paths; conservation for the former and neofunctionalization for the latter.© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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July 7, 2019

MCR-1 and OXA-48 in vivo acquisition in KPC-producing Escherichia coli after colistin treatment.

The spread of mcr-1-encoding plasmids into carbapenem-resistant Enterobacteriaceae raises concerns about the emergence of untreatable bacteria. We report the acquisition of mcr-1 in a carbapenem-resistant Escherichia coli strain after a 3-week course of colistin in a patient repatriated to France from Portugal. Whole-genome sequencing revealed that the Klebsiella pneumoniae carbapenemase-producing E. coli strain acquired two plasmids, an IncL OXA-48-encoding plasmid and an IncX4 mcr-1-encoding plasmid. This is the first report of mcr-1 in carbapenemase-encoding bacteria in France. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Complete genome sequence of super biofilm-elaborating Staphylococcus aureus isolated in Japan.

Staphylococcus aureus JP080, previously named TF2758, is a clinical isolate from an atheroma and a super biofilm-elaborating strain whose biofilm elaboration is dependent solely on polysaccharide poly-N-acetylglucosamine/polysaccharide intercellular adhesin (PNAG/PIA). Here, we report the complete genome sequence of strain JP080, which consists of one chromosome and one circular plasmid. Copyright © 2017 Yu et al.

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July 7, 2019

Insights from the genome sequence of Mycobacterium lepraemurium: Massive gene decay and reductive evolution.

Mycobacterium lepraemurium is the causative agent of murine leprosy, a chronic, granulomatous disease similar to human leprosy. Due to the similar clinical manifestations of human and murine leprosy and the difficulty of growing both bacilli axenically, Mycobacterium leprae and M. lepraemurium were once thought to be closely related, although it was later suggested that M. lepraemurium might be related to Mycobacterium avium In this study, the complete genome of M. lepraemurium was sequenced using a combination of PacBio and Illumina sequencing. Phylogenomic analyses confirmed that M. lepraemurium is a distinct species within the M. avium complex (MAC). The M. lepraemurium genome is 4.05 Mb in length, which is considerably smaller than other MAC genomes, and it comprises 2,682 functional genes and 1,139 pseudogenes, which indicates that M. lepraemurium has undergone genome reduction. An error-prone repair homologue of the DNA polymerase III a-subunit was found to be nonfunctional in M. lepraemurium, which might contribute to pseudogene formation due to the accumulation of mutations in nonessential genes. M. lepraemurium has retained the functionality of several genes thought to influence virulence among members of the MAC.IMPORTANCEMycobacterium lepraemurium seems to be evolving toward a minimal set of genes required for an obligatory intracellular lifestyle within its host, a niche seldom adopted by most mycobacteria, as they are free-living. M. lepraemurium could be used as a model to elucidate functions of genes shared with other members of the MAC. Its reduced gene set can be exploited for studying the essentiality of genes in related pathogenic species, which might lead to discovery of common virulence factors or clarify host-pathogen interactions. M. lepraemurium can be cultivated in vitro only under specific conditions and even then with difficulty. Elucidating the metabolic (in)capabilities of M. lepraemurium will help develop suitable axenic media and facilitate genetic studies. Copyright © 2017 Benjak et al.

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July 7, 2019

Genome expansion and lineage-specific genetic innovations in the forest pathogenic fungi Armillaria.

Armillaria species are both devastating forest pathogens and some of the largest terrestrial organisms on Earth. They forage for hosts and achieve immense colony sizes via rhizomorphs, root-like multicellular structures of clonal dispersal. Here, we sequenced and analysed the genomes of four Armillaria species and performed RNA sequencing and quantitative proteomic analysis on the invasive and reproductive developmental stages of A.?ostoyae. Comparison with 22 related fungi revealed a significant genome expansion in Armillaria, affecting several pathogenicity-related genes, lignocellulose-degrading enzymes and lineage-specific genes expressed during rhizomorph development. Rhizomorphs express an evolutionarily young transcriptome that shares features with the transcriptomes of both fruiting bodies and vegetative mycelia. Several genes show concomitant upregulation in rhizomorphs and fruiting bodies and share cis-regulatory signatures in their promoters, providing genetic and regulatory insights into complex multicellularity in fungi. Our results suggest that the evolution of the unique dispersal and pathogenicity mechanisms of Armillaria might have drawn upon ancestral genetic toolkits for wood-decay, morphogenesis and complex multicellularity.

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July 7, 2019

Hybrid de novo genome assembly and centromere characterization of the gray mouse lemur (Microcebus murinus).

The de novo assembly of repeat-rich mammalian genomes using only high-throughput short read sequencing data typically results in highly fragmented genome assemblies that limit downstream applications. Here, we present an iterative approach to hybrid de novo genome assembly that incorporates datasets stemming from multiple genomic technologies and methods. We used this approach to improve the gray mouse lemur (Microcebus murinus) genome from early draft status to a near chromosome-scale assembly.We used a combination of advanced genomic technologies to iteratively resolve conflicts and super-scaffold the M. murinus genome.We improved the M. murinus genome assembly to a scaffold N50 of 93.32 Mb. Whole genome alignments between our primary super-scaffolds and 23 human chromosomes revealed patterns that are congruent with historical comparative cytogenetic data, thus demonstrating the accuracy of our de novo scaffolding approach and allowing assignment of scaffolds to M. murinus chromosomes. Moreover, we utilized our independent datasets to discover and characterize sequences associated with centromeres across the mouse lemur genome. Quality assessment of the final assembly found 96% of mouse lemur canonical transcripts nearly complete, comparable to other published high-quality reference genome assemblies.We describe a new assembly of the gray mouse lemur (Microcebus murinus) genome with chromosome-scale scaffolds produced using a hybrid bioinformatic and sequencing approach. The approach is cost effective and produces superior results based on metrics of contiguity and completeness. Our results show that emerging genomic technologies can be used in combination to characterize centromeres of non-model species and to produce accurate de novo chromosome-scale genome assemblies of complex mammalian genomes.

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July 7, 2019

Draft genomes of the fungal pathogen Phellinus noxius in Hong Kong

The fungal pathogen Phellinus noxius is the underlying cause of brown root rot, a disease with causing tree mortality globally, causing extensive damage in urban areas and crop plants. This disease currently has no cure, and despite the global epidemic, little is known about the pathogenesis and virulence of this pathogen. Using Ion Torrent PGM, Illumina MiSeq and PacBio RSII sequencing platforms with various genome assembly methods, we produced the draft genome sequences of four P. noxius strains isolated from infected trees in Hong Kong to further understand the pathogen and identify the mechanisms behind the aggressive nature and virulence of this fungus. The resulting genomes ranged from 30.8Mb to 31.8Mb in size, and of the four sequences, the YTM97 strain was chosen to produce a high-quality Hong Kong strain genome sequence, resulting in a 31Mb final assembly with 457 scaffolds, an N50 length of 275,889 bp and 96.2% genome completeness. RNA-seq of YTM97 using Illumina HiSeq400 was performed for improved gene prediction. AUGUSTUS and Genemark-ES prediction programs predicted 9,887 protein-coding genes which were annotated using GO and Pfam databases. The encoded carbohydrate active enzymes revealed large numbers of lignolytic enzymes present, comparable to those of other white-rot plant pathogens. In addition, P. noxius also possessed larger numbers of cellulose, xylan and hemicellulose degrading enzymes than other plant pathogens. Searches for virulence genes was also performed using PHI-Base and DFVF databases revealing a host of virulence-related genes and effectors. The combination of non-specific host range, unique carbohydrate active enzyme profile and large amount of putative virulence genes could explain the reasons behind the aggressive nature and increased virulence of this plant pathogen. The draft genome sequences presented here will provide references for strains found in Hong Kong. Together with emerging research, this information could be used for genetic diversity and epidemiology research on a global scale as well as expediting our efforts towards discovering the mechanisms of pathogenicity of this devastating pathogen.

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July 7, 2019

Complete genome sequence of Vibrio campbellii LMB 29 isolated from red drum with four native megaplasmids.

Vibrio spp. are the most common pathogens for animals reared in aquaculture. Vibrio campbellii, which is often involved in shrimp, fish and mollusks diseases, is widely distributed in the marine environment worldwide, but our knowledge about its pathogenesis and antimicrobial resistance is very limited. The existence of this knowledge gap is at least partially because that V. campbellii was originally classified as Vibrio harveyi, and the detailed information of its comparative genome analysis to other Vibrio spp. is currently lacking. In this study, the complete genome of a V. campbellii predominant strain, LMB29, was determined by MiSeq in conjunction with PacBio SMRT sequencing. This genome consists of two circular DNA chromosomes and four megaplasmids. Comparative genome analysis indicates that LMB29 shares a 96.66% similarity (average nucleotide identity) with the V. campbellii ATCC strain BAA-1116 based on a 75% AF (average fraction) calculations, and its functional profile is very similar to V. campbellii E1 and V. campbellii CAIM115. Both type III secretion system (T3SS) and type VI secretion system (T6SS), along with the tlh gene which encodes a thermolabile hemolysin, are present in LMB29 which may contribute to the bacterial pathogenesis. The virulence of this strain was experimental confirmed by performing a LDH assay on a fish cell infection model, and cell death was observed as early as within 3 h post infection. Thirty-seven antimicrobial resistance genes (>45% identity) were predicted in LMB29 which includes a novel rifampicin ADP ribosyltransferase, arr-9, in plasmid pLMB157. The gene arr-9 was predicted on a genomic island with horizontal transferable potentials which may facilitate the rifampicin resistance dissemination. Future researches are needed to explore the pathogenesis of V. campbellii LMB29, but the availability of this genome sequence will certainly aid as a basis for further analysis.

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July 7, 2019

Global phylogenetic analysis of Escherichia coli and plasmids carrying the mcr-1 gene indicates bacterial diversity but plasmid restriction.

To understand the dynamics behind the worldwide spread of the mcr-1 gene, we determined the population structure of Escherichia coli and of mobile genetic elements (MGEs) carrying the mcr-1 gene. After a systematic review of the literature we included 65 E. coli whole genome sequences (WGS), adding 6 recently sequenced travel related isolates, and 312 MLST profiles. We included 219 MGEs described in 7 Enterobacteriaceae species isolated from human, animal and environmental samples. Despite a high overall diversity, 2 lineages were observed in the E. coli population that may function as reservoirs of the mcr-1 gene, the largest of which was linked to ST10, a sequence type known for its ubiquity in human faecal samples and in food samples. No genotypic clustering by geographical origin or isolation source was observed. Amongst a total of 13 plasmid incompatibility types, the IncI2, IncX4 and IncHI2 plasmids accounted for more than 90% of MGEs carrying the mcr-1 gene. We observed significant geographical clustering with regional spread of IncHI2 plasmids in Europe and IncI2 in Asia. These findings point towards promiscuous spread of the mcr-1 gene by efficient horizontal gene transfer dominated by a limited number of plasmid incompatibility types.

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July 7, 2019

Comparative analysis of the radish genome with Brassica genomes

Raphanus sativus L. includes an annual root vegetable crop, radish, and diverse wild species. R. sativus has a long history of domestication, but its phylogenetic position in the tribe Brassiceae is controversial. A comprehensive analysis of the R. sativus genome will provide fundamental information about the structure of its genome, evolutionary features of polyploidy, and significant insight for phylogenetic delimitation of this species. Diverse genomic resources, including a high-density genetic map, clone libraries, cytogenetic data, and transcriptome data, have been developed to sequence the genome. Recently, the R. sativus cv. ‘WK10039’ (2n = 18, 510.8 Mb) genome was sequenced and assembled into nine chromosome pseudomolecules spanning >98% of the gene space. Comparative mapping of the tPCK-like ancestral genome based on conserved ortholog set markers and proteome comparison revealed that the R. sativus genome has intermediate characteristics between the Brassica A/C and B genomes with triplicated segments, suggesting an internal origin from the genus Brassica. The evolutionary characteristics shared between R. sativus and diploid Brassica species provide genomic evidence for species delimitation of R. sativus and reconstruction of the mesohexaploid ancestral genome.

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July 7, 2019

Genetic maps and whole genome sequences of radish

Radish, Raphanus sativus L., is a member of Brassicaceae, to which Arabidopsis thaliana, a model plant in plant biology, belongs, as do other Brassica species including important crops. However, genetic and genomic studies of radish have been behind those of Arabidopsis and Brassica. In this decade, much effort has been made to develop genetic resources for radish, e.g., DNA markers, genetic maps, and whole genome sequences. Studies using the obtained information have revealed the genome structure of radish in terms of ancestral karyotype and have also prompted the identification of genes for agronomically important traits in radish through a map-based cloning strategy and quantitative trait locus analysis. In this chapter, we review the evolving development of radish genetic map in the past 15 years and the current status of genome sequencing of radish. We also introduce the latest strategy for the construction of a high-density genetic map using next-generation sequencing technology and propose a prospective direction of genetics and genomics research in radish which would be helpful for researchers and breeders in their efforts to promote radish breeding programs efficiently.

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July 7, 2019

Identification of YfiH and the catalase CatA as polyphenol oxidases of Aeromonas media and CatA as a regulator of pigmentation by Its peroxyl radical scavenging capacity.

Pyomelanin is the major constituent of pigment in melanogenic Aeromonas strains of bacteria. However, eumelanin, synthesized from tyrosine via L-DOPA and polyphenol oxidases (PPOs), may also be present in this genus since L-DOPA is frequently detected in culture fluids of several species. To address this question, we used a deletion mutant of Aeromonas media strain WS, in which pyomelanin synthesis is completely blocked under normal culture conditions. When tyrosine was supplied to the medium, we observed residual melanin accumulation, which we interpret as evidence for existence of the DOPA-melanin pathway. We traced enzymatic activity in this bacterium using native-polyacrylamide gel electrophoresis. Two PPOs: YfiH, a laccase-like protein, and CatA, a catalase, were identified. However, neither protein was critical for the residual pigmentation in pyomelanin-deficient mutant. We speculate that eumelanin synthesis may require other unknown enzymes. Deletion of yfiH did not affect pigmentation in A. media strain WS, while deletion of the CatA-encoding gene katE resulted in a reduction of melanin accumulation, but it started 9 h earlier than in the wild-type. Since catalases regulate reactive oxygen species levels during melanogenesis, we speculated that CatA affects pigmentation through its peroxyl radical scavenging capacity. Consistent with this, expression of the catalases Hpi or Hpii from Escherichia coli in the katE deletion strain of A. media strain WS restored pigmentation to the wild-type level. Hpi and Hpii also exhibited PPO activity, suggesting that catalase may represent a new class of PPOs.

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July 7, 2019

The genome sequence of Bipolaris cookei reveals mechanisms of pathogenesis underlying target leaf spot of sorghum.

Bipolaris cookei (=Bipolaris sorghicola) causes target leaf spot, one of the most prevalent foliar diseases of sorghum. Little is known about the molecular basis of pathogenesis in B. cookei, in large part due to a paucity of resources for molecular genetics, such as a reference genome. Here, a draft genome sequence of B. cookei was obtained and analyzed. A hybrid assembly strategy utilizing Illumina and Pacific Biosciences sequencing technologies produced a draft nuclear genome of 36.1?Mb, organized into 321 scaffolds with L50 of 31 and N50 of 378?kb, from which 11,189 genes were predicted. Additionally, a finished mitochondrial genome sequence of 135,790?bp was obtained, which contained 75 predicted genes. Comparative genomics revealed that B. cookei possessed substantially fewer carbohydrate-active enzymes and secreted proteins than closely related Bipolaris species. Novel genes involved in secondary metabolism, including genes implicated in ophiobolin biosynthesis, were identified. Among 37 B. cookei genes induced during sorghum infection, one encodes a putative effector with a limited taxonomic distribution among plant pathogenic fungi. The draft genome sequence of B. cookei provided novel insights into target leaf spot of sorghum and is an important resource for future investigation.

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