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Auto Tag: Genome assembly

September 22, 2019

Improved de novo genome assembly and analysis of the Chinese cucurbit Siraitia grosvenorii, also known as monk fruit or luo-han-guo.

Luo-han-guo (Siraitia grosvenorii), also called monk fruit, is a member of the Cucurbitaceae family. Monk fruit has become an important area for research because of the pharmacological and economic potential of its noncaloric, extremely sweet components (mogrosides). It is also commonly used in traditional Chinese medicine for the treatment of lung congestion, sore throat, and constipation. Recently, a single reference genome became available for monk fruit, assembled from 36.9x genome coverage reads via Illumina sequencing platforms. This genome assembly has a relatively short (34.2 kb) contig N50 length and lacks integrated annotations. These drawbacks make it difficult to use as a reference in assembling transcriptomes and discovering novel functional genes.Here, we offer a new high-quality draft of the S. grosvenorii genome assembled using 31 Gb (~73.8x) long single molecule real time sequencing reads and polished with ~50 Gb Illumina paired-end reads. The final genome assembly is approximately 469.5 Mb, with a contig N50 length of 432,384 bp, representing a 12.6-fold improvement. We further annotated 237.3 Mb of repetitive sequence and 30,565 consensus protein coding genes with combined evidence. Phylogenetic analysis showed that S. grosvenorii diverged from members of the Cucurbitaceae family approximately 40.9 million years ago. With comprehensive transcriptomic analysis and differential expression testing, we identified 4,606 up-regulated genes in the early fruit compared to the leaf, a number of which were linked to metabolic pathways regulating fruit development and ripening.The availability of this new monk fruit genome assembly, as well as the annotations, will facilitate the discovery of new functional genes and the genetic improvement of monk fruit.

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September 22, 2019

Transcriptional regulation of cysteine and methionine metabolism in Lactobacillus paracasei FAM18149.

Lactobacillus paracasei is common in the non-starter lactic acid bacteria (LAB) community of raw milk cheeses. This species can significantly contribute to flavor formation through amino acid metabolism. In this study, the DNA and RNA of L. paracasei FAM18149 were sequenced using next-generation sequencing technologies to reconstruct the metabolism of the sulfur-containing amino acids cysteine and methionine. Twenty-three genes were found to be involved in cysteine biosynthesis, the conversion of cysteine to methionine and vice versa, the S-adenosylmethionine recycling pathway, and the transport of sulfur-containing amino acids. Additionally, six methionine-specific T-boxes and one cysteine-specific T-box were found. Five of these were located upstream of genes encoding transporter functions. RNA-seq analysis and reverse-transcription quantitative polymerase reaction assays showed that expression of genes located downstream of these T-boxes was affected by the absence of either cysteine or methionine. Remarkably, the cysK2-ctl1-cysE2 operon, which is associated with te methionine-to-cysteine conversion and is upregulated in the absence of cysteine, showed high read coverage in the 5′-untranslated region and an antisense-RNA in the 3′-untranslated region. This indicates that this operon is regulated by the combination of cis- and antisense-mediated regulation mechanisms. The results of this study may help in the selection of L. paracasei strains to control sulfuric flavor formation in cheese.

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September 22, 2019

Co-culture of soil biofilm isolates enables the discovery of novel antibiotics

Bacterial natural products (NPs) are considered to be a promising source of drug discovery. However, the biosynthesis gene clusters (BGCs) of NP are not often expressed, making it difficult to identify them. Recently, the study of biofilm community showed bacteria may gain competitive advantages by the secretion of antibiotics, implying a possible way to screen antibiotic by evaluating the social behavior of bacteria. In this study, we have described an efficient workflow for novel antibiotic discovery by employing the bacterial social interaction strategy with biofilm cultivation, co-culture, transcriptomic and genomic methods. We showed that a biofilm dominant species, i.e. Pseudomonas sp. G7, which was isolated from cultivated soil biofilm community, was highly competitive in four-species biofilm communities, as the synergistic combinations preferred to exclude this strain while the antagonistic combinations did not. Through the analysis of transcriptomic changes in four-species co-culture and the complete genome of Pseudomonas sp. G7, we finally discovered two novel non-ribosomal polypeptide synthetic (NRPS) BGCs, whose products were predicted to have seven and six amino acid components, respectively. Furthermore, we provide evidence showing that only when Pseudomonas sp. G7 was co-cultivated with at least two or three other bacterial species can these BGC genes be induced, suggesting that the co-culture of the soil biofilm isolates is critical to the discovery of novel antibiotics. As a conclusion, we set a model of applying microbial interaction to the discovery of new antibiotics.

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September 22, 2019

Diversity of hepatitis E virus genotype 3

Summary Hepatitis E virus genotype 3 (HEV-3) can lead to chronic infection in immunocompromised patients, and ribavirin is the treatment of choice. Recently, mutations in the polymerase gene have been associated with ribavirin failure but their frequency before treatment according to HEV-3 subtypes has not been studied on a large data set. We used single-molecule real-time sequencing technology to sequence 115 new complete genomes of HEV-3 infecting French patients. We analyzed phylogenetic relationships, the length of the polyproline region, and mutations in the HEV polymerase gene. Eighty-five (74%) were in the clade HEV-3efg, 28 (24%) in HEV-3chi clade, and 2 (2%) in HEV-3ra clade. Using automated partitioning of maximum likelihood phylogenetic trees, complete genomes were classified into subtypes. Polyproline region length differs within HEV-3 clades (from 189 to 315 nt). Investigating mutations in the polymerase gene, distinct polymorphisms between HEV-3 subtypes were found (G1634R in 95% of HEV-3e, G1634K in 56% of HEV-3ra, and V1479I in all HEV-3efg, clade HEV-3ra, and HEV-3k strains). Subtype-specific polymorphisms in the HEV-3 polymerase have been identified. Our study provides new complete genome sequences of HEV-3 that could be useful for comparing strains circulating in humans and the animal reservoir.

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September 22, 2019

De novo genome assembly of Oryza granulata reveals rapid genome expansion and adaptive evolution

The wild relatives of rice have adapted to different ecological environments and constitute a useful reservoir of agronomic traits for genetic improvement. Here we present the ~777?Mb de novo assembled genome sequence of Oryza granulata. Recent bursts of long-terminal repeat retrotransposons, especially RIRE2, led to a rapid twofold increase in genome size after O. granulata speciation. Universal centromeric tandem repeats are absent within its centromeres, while gypsy-type LTRs constitute the main centromere-specific repetitive elements. A total of 40,116 protein-coding genes were predicted in O. granulata, which is close to that of Oryza sativa. Both the copy number and function of genes involved in photosynthesis and energy production have undergone positive selection during the evolution of O. granulata, which might have facilitated its adaptation to the low light habitats. Together, our findings reveal the rapid genome expansion, distinctive centromere organization, and adaptive evolution of O. granulata.

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September 22, 2019

HapCHAT: adaptive haplotype assembly for efficiently leveraging high coverage in long reads.

Haplotype assembly is the process of assigning the different alleles of the variants covered by mapped sequencing reads to the two haplotypes of the genome of a human individual. Long reads, which are nowadays cheaper to produce and more widely available than ever before, have been used to reduce the fragmentation of the assembled haplotypes since their ability to span several variants along the genome. These long reads are also characterized by a high error rate, an issue which may be mitigated, however, with larger sets of reads, when this error rate is uniform across genome positions. Unfortunately, current state-of-the-art dynamic programming approaches designed for long reads deal only with limited coverages.Here, we propose a new method for assembling haplotypes which combines and extends the features of previous approaches to deal with long reads and higher coverages. In particular, our algorithm is able to dynamically adapt the estimated number of errors at each variant site, while minimizing the total number of error corrections necessary for finding a feasible solution. This allows our method to significantly reduce the required computational resources, allowing to consider datasets composed of higher coverages. The algorithm has been implemented in a freely available tool, HapCHAT: Haplotype Assembly Coverage Handling by Adapting Thresholds. An experimental analysis on sequencing reads with up to 60 × coverage reveals improvements in accuracy and recall achieved by considering a higher coverage with lower runtimes.Our method leverages the long-range information of sequencing reads that allows to obtain assembled haplotypes fragmented in a lower number of unphased haplotype blocks. At the same time, our method is also able to deal with higher coverages to better correct the errors in the original reads and to obtain more accurate haplotypes as a result.HapCHAT is available at http://hapchat.algolab.eu under the GNU Public License (GPL).

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September 22, 2019

Raising the stakes: Loss of efflux pump regulation decreases meropenem susceptibility in Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of the high-mortality disease melioidosis, is a gram-negative bacterium that is naturally resistant to many antibiotics. There is no vaccine for melioidosis, and effective eradication is reliant on biphasic and prolonged antibiotic administration. The carbapenem drug meropenem is the current gold standard option for treating severe melioidosis. Intrinsic B. pseudomallei resistance toward meropenem has not yet been documented; however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and treatment failure.We examined our 30-year clinical collection of melioidosis cases to identify B. pseudomallei isolates with reduced meropenem susceptibility. Isolates were subjected to minimum inhibitory concentration (MIC) testing toward meropenem. Paired isolates from patients who had evolved decreased susceptibility were subjected to whole-genome sequencing. Select agent-compliant genetic manipulation was carried out to confirm the molecular mechanisms conferring resistance.We identified 11 melioidosis cases where B. pseudomallei isolates developed decreased susceptibility toward meropenem during treatment, including 2 cases not treated with this antibiotic. Meropenem MICs increased from 0.5-0.75 µg/mL to 3-8 µg/mL. Comparative genomics identified multiple mutations affecting multidrug resistance-nodulation-division (RND) efflux pump regulators, with concomitant overexpression of their corresponding pumps. All cases were refractory to treatment despite aggressive, targeted therapy, and 2 were associated with a fatal outcome.This study confirms the role of RND efflux pumps in decreased meropenem susceptibility in B. pseudomallei. These findings have important ramifications for the diagnosis, treatment, and management of life-threatening melioidosis cases.

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September 22, 2019

Comparative genomics of Pseudomonas syringae reveals convergent gene gain and loss associated with specialization onto cherry (Prunus avium).

Genome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium), including pathovars P. syringae pv morsprunorum (Psm) races 1 and 2, P. syringae pv syringae (Pss) and P. syringae pv avii. Phylogenetic analyses revealed Psm races and P. syringae pv avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species. A maximum likelihood approach was used to predict effectors associated with pathogenicity on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters than Psm and P. syringae pv avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions. Cherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host.© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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September 22, 2019

Comparative genomics of Spiraeoideae-infecting Erwinia amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low-virulence strain.

Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae-infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations (‘Widely Prevalent’ clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low-virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.© 2017 BSPP AND JOHN WILEY & SONS LTD.

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September 22, 2019

RAD sequencing and a hybrid Antarctic fur seal genome assembly reveal rapidly decaying linkage disequilibrium, global population structure and evidence for inbreeding.

Recent advances in high throughput sequencing have transformed the study of wild organisms by facilitating the generation of high quality genome assemblies and dense genetic marker datasets. These resources have the potential to significantly advance our understanding of diverse phenomena at the level of species, populations and individuals, ranging from patterns of synteny through rates of linkage disequilibrium (LD) decay and population structure to individual inbreeding. Consequently, we used PacBio sequencing to refine an existing Antarctic fur seal (Arctocephalus gazella) genome assembly and genotyped 83 individuals from six populations using restriction site associated DNA (RAD) sequencing. The resulting hybrid genome comprised 6,169 scaffolds with an N50 of 6.21 Mb and provided clear evidence for the conservation of large chromosomal segments between the fur seal and dog (Canis lupus familiaris). Focusing on the most extensively sampled population of South Georgia, we found that LD decayed rapidly, reaching the background level by around 400 kb, consistent with other vertebrates but at odds with the notion that fur seals experienced a strong historical bottleneck. We also found evidence for population structuring, with four main Antarctic island groups being resolved. Finally, appreciable variance in individual inbreeding could be detected, reflecting the strong polygyny and site fidelity of the species. Overall, our study contributes important resources for future genomic studies of fur seals and other pinnipeds while also providing a clear example of how high throughput sequencing can generate diverse biological insights at multiple levels of organization. Copyright © 2018 Humble et al.

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September 22, 2019

Whole-genome sequence and genome annotation of Xanthomonas citri pv. mangiferaeindicae, causal agent of bacterial black spot on Mangifera indica.

A newly isolated strain XC01 was identified as Xanthomonas citri pv. mangiferaeindicae, isolated from an infected mango fruit in Guangxi, China. The complete genome sequence of XC01 was carried out using the PacBio RSII platform. The genome contains a circular chromosome with 3,865,165 bp, 3442 protein-coding genes, 53 tRNAs, and 2 rRNA operons. Phylogenetic analysis revealed that this pathogen is very close to the soybeans bacterial pustule pathogen X. citri pv. glycines CFBP 2526, with a completely different host range. The genome sequence of XC01 may shed a highlight genes with a demonstrated or proposed role in on the pathogenesis.

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September 22, 2019

Whole-genome comparison of high and low virulent Staphylococcus aureus isolates inducing implant-associated bone infections.

Staphylococcus aureus can cause wide range of infections from simple soft skin infections to severe endocarditis, bacteremia, osteomyelitis and implant associated bone infections (IABI). The focus of the present investigation was to study virulence properties of S. aureus isolates from acute and chronic IABI by means of their in vivo lethality, in vitro osteoblasts invasion, biofilm formation and subsequently whole genome comparison between high and low virulent strains. Application of insect infection model Galleria mellonella revealed high, intermediate and low virulence phenotypes of these clinical isolates, which showed good correlation with osteoblast invasion and biofilm formation assays. Comparative genomics of selected high (EDCC 5458) and low (EDCC 5464) virulent strains enabled the identification of molecular factors responsible for the development of acute and chronic IABI. Accordingly, the low virulent strain EDCC 5464 harbored point mutations resulting in frame shift mutations in agrC (histidine kinase in agr system), graS (histidine kinase in graSR, a two component system) and efeB (peroxidase in efeOBU operon, an iron acquisition system) genes. Additionally, we found a mobile element (present 11 copies in EDCC 5464) inserted at the end of ß-hemolysin (hlb) and sarU genes, which are involved in the pathogenesis and regulation of virulence gene expression in coordination with quorum sensing system. All these results are in good support with the low virulence behavior of EDCC 5464. From the previous literature, it is well known that agr defective S. aureus clinical strains are isolated from the chronic infections. Similarly, low virulent EDCC 5464 was isolated from chronic implant-associated bone infections infection whereas EDCC 5458 was obtained from acute implant-associated bone infections. Laboratory based in vitro and in vivo results and insights from comparative genomic analysis could be correlated with the clinical conclusion of IABIs and allows evidence-based treatment strategies based on the pathogenesis of the strain to cure life devastating implant-associated infections. Copyright © 2018 Elsevier GmbH. All rights reserved.

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September 22, 2019

Investigating the central metabolism of Clostridium thermosuccinogenes.

Clostridium thermosuccinogenes is a thermophilic anaerobic bacterium able to convert various carbohydrates to succinate and acetate as main fermentation products. Genomes of the four publicly available strains have been sequenced, and the genome of the type strain has been closed. The annotated genomes were used to reconstruct the central metabolism, and enzyme assays were used to validate annotations and to determine cofactor specificity. The genes were identified for the pathways to all fermentation products, as well as for the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway. Notably, a candidate transaldolase was lacking, and transcriptomics during growth on glucose versus that on xylose did not provide any leads to potential transaldolase genes or alternative pathways connecting the C5 with the C3/C6 metabolism. Enzyme assays showed xylulokinase to prefer GTP over ATP, which could be of importance for engineering xylose utilization in related thermophilic species of industrial relevance. Furthermore, the gene responsible for malate dehydrogenase was identified via heterologous expression in Escherichia coli and subsequent assays with the cell extract, which has proven to be a simple and powerful method for the basal characterization of thermophilic enzymes.IMPORTANCE Running industrial fermentation processes at elevated temperatures has several advantages, including reduced cooling requirements, increased reaction rates and solubilities, and a possibility to perform simultaneous saccharification and fermentation of a pretreated biomass. Most studies with thermophiles so far have focused on bioethanol production. Clostridium thermosuccinogenes seems an attractive production organism for organic acids, succinic acid in particular, from lignocellulosic biomass-derived sugars. This study provides valuable insights into its central metabolism and GTP and PPi cofactor utilization. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Clonal emergence of invasive multidrug-resistant Staphylococcus epidermidis deconvoluted via a combination of whole-genome sequencing and microbiome analyses.

Pathobionts, bacteria that are typically human commensals but can cause disease, contribute significantly to antimicrobial resistance. Staphylococcus epidermidis is a prototypical pathobiont as it is a ubiquitous human commensal but also a leading cause of healthcare-associated bacteremia. We sought to determine the etiology of a recent increase in invasive S. epidermidis isolates resistant to linezolid.Whole-genome sequencing (WGS) was performed on 176 S. epidermidis bloodstream isolates collected at the MD Anderson Cancer Center in Houston, Texas, between 2013 and 2016. Molecular relationships were assessed via complementary phylogenomic approaches. Abundance of the linezolid resistance determinant cfr was determined in stool samples via reverse-transcription quantitative polymerase chain reaction.Thirty-nine of the 176 strains were linezolid resistant (22%). Thirty-one of the 39 linezolid-resistant S. epidermidis infections were caused by a particular clone resistant to multiple antimicrobials that spread among leukemia patients and carried cfr on a 49-kb plasmid (herein called pMB151a). The 6 kb of pMB151a surrounding the cfr gene was nearly 100% identical to a cfr-containing plasmid isolated from livestock-associated staphylococci in China. Analysis of serial stool samples from leukemia patients revealed progressive staphylococcal domination of the intestinal microflora and an increase in cfr abundance following linezolid use.The combination of linezolid use plus transmission of a multidrug-resistant clone drove expansion of invasive, linezolid-resistant S. epidermidis. Our results lend support to the notion that a combination of antibiotic stewardship plus infection control measures may help to control the spread of a multidrug-resistant pathobiont.

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September 22, 2019

Redefinition and unification of the SXT/R391 family of integrative and conjugative elements.

Integrative and conjugative elements (ICEs) of the SXT/R391 family are key drivers of the spread of antibiotic resistance in Vibrio cholerae, the infectious agent of cholera, and other pathogenic bacteria. The SXT/R391 family of ICEs was defined based on the conservation of a core set of 52 genes and site-specific integration into the 5′ end of the chromosomal gene prfC Hence, the integrase gene int has been intensively used as a marker to detect SXT/R391 ICEs in clinical isolates. ICEs sharing most core genes but differing by their integration site and integrase gene have been recently reported and excluded from the SXT/R391 family. Here we explored the prevalence and diversity of atypical ICEs in GenBank databases and their relationship with typical SXT/R391 ICEs. We found atypical ICEs in V. cholerae isolates that predate the emergence and expansion of typical SXT/R391 ICEs in the mid-1980s in seventh-pandemic toxigenic V. cholerae strains O1 and O139. Our analyses revealed that while atypical ICEs are not associated with antibiotic resistance genes, they often carry cation efflux pumps, suggesting heavy metal resistance. Atypical ICEs constitute a polyphyletic group likely because of occasional recombination events with typical ICEs. Furthermore, we show that the alternative integration and excision genes of atypical ICEs remain under the control of SetCD, the main activator of the conjugative functions of SXT/R391 ICEs. Together, these observations indicate that substitution of the integration/excision module and change of specificity of integration do not preclude atypical ICEs from inclusion into the SXT/R391 family.IMPORTANCEVibrio cholerae is the causative agent of cholera, an acute intestinal infection that remains to this day a world public health threat. Integrative and conjugative elements (ICEs) of the SXT/R391 family have played a major role in spreading antimicrobial resistance in seventh-pandemic V. cholerae but also in several species of Enterobacteriaceae Most epidemiological surveys use the integrase gene as a marker to screen for SXT/R391 ICEs in clinical or environmental strains. With the recent reports of closely related elements that carry an alternative integrase gene, it became urgent to investigate whether ICEs that have been left out of the family are a liability for the accuracy of such screenings. In this study, based on comparative genomics, we broaden the SXT/R391 family of ICEs to include atypical ICEs that are often associated with heavy metal resistance. Copyright © 2018 American Society for Microbiology.

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