Menu

Auto Tag: Validation

September 22, 2019

Comparative genomics reveals cotton-specific virulence factors in flexible genomic regions in Verticillium dahliae and evidence of horizontal gene transfer from Fusarium.

Verticillium dahliae isolates are most virulent on the host from which they were originally isolated. Mechanisms underlying these dominant host adaptations are currently unknown. We sequenced the genome of V. dahliae Vd991, which is highly virulent on its original host, cotton, and performed comparisons with the reference genomes of JR2 (from tomato) and VdLs.17 (from lettuce). Pathogenicity-related factor prediction, orthology and multigene family classification, transcriptome analyses, phylogenetic analyses, and pathogenicity experiments were performed. The Vd991 genome harbored several exclusive, lineage-specific (LS) genes within LS regions (LSRs). Deletion mutants of the seven genes within one LSR (G-LSR2) in Vd991 were less virulent only on cotton. Integration of G-LSR2 genes individually into JR2 and VdLs.17 resulted in significantly enhanced virulence on cotton but did not affect virulence on tomato or lettuce. Transcription levels of the seven LS genes in Vd991 were higher during the early stages of cotton infection, as compared with other hosts. Phylogenetic analyses suggested that G-LSR2 was acquired from Fusarium oxysporum f. sp. vasinfectum through horizontal gene transfer. Our results provide evidence that horizontal gene transfer from Fusarium to Vd991 contributed significantly to its adaptation to cotton and may represent a significant mechanism in the evolution of an asexual plant pathogen.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Read more
September 22, 2019

Reduction of nonspecificity motifs in synthetic antibody libraries.

Successful antibody development requires both functional binding and desirable biophysical characteristics. In the current study, we analyze the causes of one hurdle to clinical development, off-target reactivity, or nonspecificity. We used a high-throughput nonspecificity assay to isolate panels of nonspecific antibodies from two synthetic single-chain variable fragment libraries expressed on the surface of yeast, identifying both individual amino acids and motifs within the complementarity-determining regions which contribute to the phenotype. We find enrichment of glycine, valine, and arginine as both individual amino acids and as a part of motifs, and additionally enrichment of motifs containing tryptophan. Insertion of any of these motifs into the complementarity-determining region H3 of a “clean” antibody increased its nonspecificity, with greatest increases in antibodies containing Trp or Val motifs. We next applied these rules to the creation of a synthetic diversity library based on natural frameworks with significantly decreased incorporation of such motifs and demonstrated its ability to isolate binders to a wide panel of antigens. This work both provides a greater understanding of the drivers of nonspecificity and provides design rules to increase efficiency in the isolation of antibodies with drug-like properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

Read more
September 22, 2019

Translating genomics into practice for real-time surveillance and response to carbapenemase-producing Enterobacteriaceae: evidence from a complex multi-institutional KPC outbreak.

Until recently, Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks.All KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each isolate. This was complemented with PacBio long-read sequencing on selected isolates to establish high-quality reference sequences and interrogate characteristics of KPC-encoding plasmids.Initial investigations indicated that the outbreak was widespread, with 86 KPC-producing Enterobacteriaceae isolates (K. pneumoniae 92%) identified from 35 different locations across metropolitan and rural Victoria between 2012 and 2015. Initial combined analyses of the epidemiological and genomic data resolved the outbreak into distinct nosocomial transmission networks, and identified healthcare facilities at the epicentre of KPC transmission. New cases were assigned to transmission networks in real-time, allowing focussed infection control efforts. PacBio sequencing confirmed a secondary transmission network arising from inter-species plasmid transmission. Insights from Bayesian transmission inference and analyses of within-host diversity informed the development of state-wide public health and infection control guidelines, including interventions such as an intensive approach to screening contacts following new case detection to minimise unrecognised colonisation.A real-time combined epidemiological and genomic investigation proved critical to identifying and defining multiple transmission networks of KPC Enterobacteriaceae, while data from either investigation alone were inconclusive. The investigation was fundamental to informing infection control measures in real-time and the development of state-wide public health guidelines on carbapenemase-producing Enterobacteriaceae surveillance and management.

Read more
September 22, 2019

The genomes of Crithidia bombi and C. expoeki, common parasites of bumblebees.

Trypanosomatids (Trypanosomatidae, Kinetoplastida) are flagellated protozoa containing many parasites of medical or agricultural importance. Among those, Crithidia bombi and C. expoeki, are common parasites in bumble bees around the world, and phylogenetically close to Leishmania and Leptomonas. They have a simple and direct life cycle with one host, and partially castrate the founding queens greatly reducing their fitness. Here, we report the nuclear genome sequences of one clone of each species, extracted from a field-collected infection. Using a combination of Roche 454 FLX Titanium, Pacific Biosciences PacBio RS, and Illumina GA2 instruments for C. bombi, and PacBio for C. expoeki, we could produce high-quality and well resolved sequences. We find that these genomes are around 32 and 34 MB, with 7,808 and 7,851 annotated genes for C. bombi and C. expoeki, respectively-which is somewhat less than reported from other trypanosomatids, with few introns, and organized in polycistronic units. A large fraction of genes received plausible functional support in comparison primarily with Leishmania and Trypanosoma. Comparing the annotated genes of the two species with those of six other trypanosomatids (C. fasciculata, L. pyrrhocoris, L. seymouri, B. ayalai, L. major, and T. brucei) shows similar gene repertoires and many orthologs. Similar to other trypanosomatids, we also find signs of concerted evolution in genes putatively involved in the interaction with the host, a high degree of synteny between C. bombi and C. expoeki, and considerable overlap with several other species in the set. A total of 86 orthologous gene groups show signatures of positive selection in the branch leading to the two Crithidia under study, mostly of unknown function. As an example, we examined the initiating glycosylation pathway of surface components in C. bombi, finding it deviates from most other eukaryotes and also from other kinetoplastids, which may indicate rapid evolution in the extracellular matrix that is involved in interactions with the host. Bumble bees are important pollinators and Crithidia-infections are suspected to cause substantial selection pressure on their host populations. These newly sequenced genomes provide tools that should help better understand host-parasite interactions in these pollinator pathogens.

Read more
September 22, 2019

Functional metagenomics reveals a novel carbapenem-hydrolyzing mobile beta-lactamase from Indian river sediments contaminated with antibiotic production waste.

Evolution has provided environmental bacteria with a plethora of genes that give resistance to antibiotic compounds. Under anthropogenic selection pressures, some of these genes are believed to be recruited over time into pathogens by horizontal gene transfer. River sediment polluted with fluoroquinolones and other drugs discharged from bulk drug production in India constitute an environment with unprecedented, long-term antibiotic selection pressures. It is therefore plausible that previously unknown resistance genes have evolved and/or are promoted here. In order to search for novel resistance genes, we therefore analyzed such river sediments by a functional metagenomics approach. DNA fragments providing resistance to different antibiotics in E. coli were sequenced using Sanger and PacBio RSII platforms. We recaptured the majority of known antibiotic resistance genes previously identified by open shot-gun metagenomics sequencing of the same samples. In addition, seven novel resistance gene candidates (six beta-lactamases and one amikacin resistance gene) were identified. Two class A beta-lactamases, blaRSA1 and blaRSA2, were phylogenetically close to clinically important ESBLs like blaGES, blaBEL and blaL2, and were further characterized for their substrate spectra. The blaRSA1 protein, encoded as an integron gene cassette, efficiently hydrolysed penicillins, first generation cephalosporins and cefotaxime, while blaRSA2 was an inducible class A beta-lactamase, capable of hydrolyzing carbapenems albeit with limited efficiency, similar to the L2 beta-lactamase from Stenotrophomonas maltophilia. All detected novel genes were associated with plasmid mobilization proteins, integrons, and/or other resistance genes, suggesting a potential for mobility. This study provides insight into a resistome shaped by an exceptionally strong and long-term antibiotic selection pressure. An improved knowledge of mobilized resistance factors in the external environment may make us better prepared for the resistance challenges that we may face in clinics in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Read more
September 22, 2019

SimulaTE: simulating complex landscapes of transposable elements of populations.

Motivation Estimating the abundance of transposable elements (TEs) in populations (or tissues) promises to answer many open research questions. However, progress is hampered by the lack of concordance between different approaches for TE identification and thus potentially unreliable results. Results To address this problem, we developed SimulaTE a tool that generates TE landscapes for populations using a newly developed domain specific language (DSL). The simple syntax of our DSL allows for easily building even complex TE landscapes that have, for example, nested, truncated and highly diverged TE insertions. Reads may be simulated for the populations using different sequencing technologies (PacBio, Illumina paired-ends) and strategies (sequencing individuals and pooled populations). The comparison between the expected (i.e. simulated) and the observed results will guide researchers in finding the most suitable approach for a particular research question. Availability and implementation SimulaTE is implemented in Python and available at https://sourceforge.net/projects/simulates/. Manual https://sourceforge.net/p/simulates/wiki/Home/#manual; Test data and tutorials https://sourceforge.net/p/simulates/wiki/Home/#walkthrough; Validation https://sourceforge.net/p/simulates/wiki/Home/#validation. Contact robert.kofler@vetmeduni.ac.at

Read more
September 22, 2019

The draft genome assembly of Dermatophagoides pteronyssinus supports identification of novel allergen isoforms in Dermatophagoides species.

Background: Dermatophagoides pteronyssinus (DP) and Dermatophagoides farinae (DF) are highly similar disease-asso- ciated mites with frequently overlapping geographic distributions. A draft genome of DP was assembled to identify the candidate allergens in DP that are homologous to those in DF, investigate allergen isoforms, and facilitate comparisons with related Acari. Methods: PacBio and Illumina whole-genome sequencing was performed on DP. Assembly and reconstruction of the genomes were optimized for isoform identification in a heterogeneous population. Bioinformatic analyses of Acari genomes were performed. Results: The predicted size of the DP nuclear genome is 52.5 Mb. A predicted set of 19,368 proteins was identified, including all 19 currently recognized allergens from this species. Orthologs for 12 allergens established for DF were found. The population of DP mites showed a high level of heterozygosity that allowed the identification of 43 new isoforms for both established and candidate allergens in DP including a new isoform for the major allergen Der p 23. Reanalyzing the previous DF data assuming heterozygosity, 14 new allergen isoforms could be identified. Some new isoforms were observed in both species, suggesting that these isoforms predated speciation. The high quality of both genomes allowed an examination of synteny which showed that many allergen orthologs are physically clustered but with species-specific exon/intron structures. Comparative genomic analyses of other Acariformes mites showed that most of the allergen homologs are widely conserved within this Superorder. Conclusions: Candidate allergens in DP were identified to facilitate future serological studies. While DP and DF are highly similar genetically, species-specific allergen isoforms exist to facilitate molecular differentiation.

Read more
September 22, 2019

Identification of candidate genes at the Dp-fl locus conferring resistance against the rosy apple aphid Dysaphis plantaginea

The cultivated apple is susceptible to several pests including the rosy apple aphid (RAA; Dysaphis plantaginea Passerini), control of which is mainly based on chemical treatments. A few cases of resistance to aphids have been described in apple germplasm resources, laying the basis for the development of new resistant cultivars by breeding. The cultivar ‘Florina’ is resistant to RAA, and recently, the Dp-fl locus responsible for its resistance was mapped on linkage group 8 of the apple genome. In this paper, a chromosome walking approach was performed by using a ‘Florina’ bacterial artificial chromosome (BAC) library. The walking started from the available tightly linked molecular markers flanking the resistance region. Various walking steps were performed in order to identify the minimum tiling path of BAC clones covering the Dp-fl region from both the “resistant” and “susceptible” chromosomes of ‘Florina’. A genomic region of about 279 Kb encompassing the Dp-fl resistance locus was fully sequenced by the PacBio technology. Through the development of new polymorphic markers, the mapping interval around the resistance locus was narrowed down to a physical region of 95 Kb. The annotation of this sequence resulted in the identification of four candidate genes putatively involved in the RAA resistance response.

Read more
September 22, 2019

Characterization of plasmids harboring blaCTX-M and blaCMY genes in E. coli from French broilers.

Resistance to extended-spectrum cephalosporins (ESC) is a global health issue. The aim of this study was to analyze and compare plasmids coding for resistance to ESC isolated from 16 avian commensal and 17 avian pathogenic Escherichia coli (APEC) strains obtained respectively at slaughterhouse or from diseased broilers in 2010-2012. Plasmid DNA was used to transform E. coli DH5alpha, and the resistances of the transformants were determined. The sequences of the ESC-resistance plasmids prepared from transformants were obtained by Illumina (33 plasmids) or PacBio (1 plasmid). Results showed that 29 of these plasmids contained the blaCTX-M-1 gene and belonged to the IncI1/ST3 type, with 27 and 20 of them carrying the sul2 or tet(A) genes respectively. Despite their diverse origins, several plasmids showed very high percentages of identity. None of the blaCTX-M-1-containing plasmid contained APEC virulence genes, although some of them were detected in the parental strains. Three plasmids had the blaCMY-2 gene, but no other resistance gene. They belonged to IncB/O/K/Z-like or IncFIA/FIB replicon types. The blaCMY-2 IncFIA/FIB plasmid was obtained from a strain isolated from a diseased broiler and also containing a blaCTX-M-1 IncI1/ST3 plasmid. Importantly APEC virulence genes (sitA-D, iucA-D, iutA, hlyF, ompT, etsA-C, iss, iroB-E, iroN, cvaA-C and cvi) were detected on the blaCMY-2 plasmid. In conclusion, our results show the dominance and high similarity of blaCTX-M-1 IncI1/ST3 plasmids, and the worrying presence of APEC virulence genes on a blaCMY-2 plasmid.

Read more
September 22, 2019

Redkmer: An Assembly-Free Pipeline for the Identification of Abundant and Specific X-Chromosome Target Sequences for X-Shredding by CRISPR Endonucleases.

CRISPR-based synthetic sex ratio distorters, which operate by shredding the X-chromosome during male meiosis, are promising tools for the area-wide control of harmful insect pest or disease vector species. X-shredders have been proposed as tools to suppress insect populations by biasing the sex ratio of the wild population toward males, thus reducing its natural reproductive potential. However, to build synthetic X-shredders based on CRISPR, the selection of gRNA targets, in the form of high-copy sequence repeats on the X chromosome of a given species, is difficult, since such repeats are not accurately resolved in genome assemblies and cannot be assigned to chromosomes with confidence. We have therefore developed the redkmer computational pipeline, designed to identify short and highly abundant sequence elements occurring uniquely on the X chromosome. Redkmer was designed to use as input minimally processed whole genome sequence data from males and females. We tested redkmer with short- and long-read whole genome sequence data of Anopheles gambiae, the major vector of human malaria, in which the X-shredding paradigm was originally developed. Redkmer established long reads as chromosomal proxies with excellent correlation to the genome assembly and used them to rank X-candidate kmers for their level of X-specificity and abundance. Among these, a high-confidence set of 25-mers was identified, many belonging to previously known X-chromosome repeats of Anopheles gambiae, including the ribosomal gene array and the selfish elements harbored within it. Data from a control strain, in which these repeats are shared with the Y chromosome, confirmed the elimination of these kmers during filtering. Finally, we show that redkmer output can be linked directly to gRNA selection and off-target prediction. In addition, the output of redkmer, including the prediction of chromosomal origin of single-molecule long reads and chromosome specific kmers, could also be used for the characterization of other biologically relevant sex chromosome sequences, a task that is frequently hampered by the repetitiveness of sex chromosome sequence content.

Read more
September 22, 2019

Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza.

The genus Oryza is a model system for the study of molecular evolution over time scales ranging from a few thousand to 15 million years. Using 13 reference genomes spanning the Oryza species tree, we show that despite few large-scale chromosomal rearrangements rapid species diversification is mirrored by lineage-specific emergence and turnover of many novel elements, including transposons, and potential new coding and noncoding genes. Our study resolves controversial areas of the Oryza phylogeny, showing a complex history of introgression among different chromosomes in the young ‘AA’ subclade containing the two domesticated species. This study highlights the prevalence of functionally coupled disease resistance genes and identifies many new haplotypes of potential use for future crop protection. Finally, this study marks a milestone in modern rice research with the release of a complete long-read assembly of IR 8 ‘Miracle Rice’, which relieved famine and drove the Green Revolution in Asia 50 years ago.

Read more
September 22, 2019

The putative functions of lysogeny in mediating the survivorship of Escherichia coli in seawater and marine sediment.

Escherichia coli colonizes various body parts of animal hosts as a commensal and a pathogen. It can also persist in the external environment in the absence of fecal pollution. It remains unclear how this species has evolved to adapt to such contrasting habitats. Lysogeny plays pivotal roles in the diversification of the phenotypic and ecologic characters of E. coli as a symbiont. We hypothesized that lysogeny could also confer fitness to survival in the external environment. To test this hypothesis, we used the induced phages of an E. coli strain originating from marine sediment to infect a fecal E. coli strain to obtain an isogenic lysogen of the latter. The three strains were tested for survivorship in microcosms of seawater, marine sediment and sediment interstitial water as well as swimming motility, glycogen accumulation, biofilm formation, substrate utilization and stress resistance. The results indicate that lysogenic infection led to tractable changes in many of the ecophysiological attributes tested. Particularly, the lysogen had better survivorship in the microcosms and had a substrate utilization profile resembling the sediment strain more than the wild type fecal strain. Our findings provide new insights into the understanding of how E. coli survives in the natural environment.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Read more
September 22, 2019

Comparative genomics reveals new single-nucleotide polymorphisms that can assist in identification of adherent-invasive Escherichia coli.

Adherent-invasive Escherichia coli (AIEC) have been involved in Crohn’s disease (CD). Currently, AIEC are identified by time-consuming techniques based on in vitro infection of cell lines to determine their ability to adhere to and invade intestinal epithelial cells as well as to survive and replicate within macrophages. Our aim was to find signature sequences that can be used to identify the AIEC pathotype. Comparative genomics was performed between three E. coli strain pairs, each pair comprised one AIEC and one non-AIEC with identical pulsotype, sequence type and virulence gene carriage. Genetic differences were further analysed in 22 AIEC and 28 non-AIEC isolated from CD patients and controls. The strain pairs showed similar genome structures, and no gene was specific to AIEC. Three single nucleotide polymorphisms displayed different nucleotide distributions between AIEC and non-AIEC, and four correlated with increased adhesion and/or invasion indices. Here, we present a classification algorithm based on the identification of three allelic variants that can predict the AIEC phenotype with 84% accuracy. Our study corroborates the absence of an AIEC-specific genetic marker distributed across all AIEC strains. Nonetheless, point mutations putatively involved in the AIEC phenotype can be used for the molecular identification of the AIEC pathotype.

Read more
September 22, 2019

The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution.

The sea lamprey (Petromyzon marinus) serves as a comparative model for reconstructing vertebrate evolution. To enable more informed analyses, we developed a new assembly of the lamprey germline genome that integrates several complementary data sets. Analysis of this highly contiguous (chromosome-scale) assembly shows that both chromosomal and whole-genome duplications have played significant roles in the evolution of ancestral vertebrate and lamprey genomes, including chromosomes that carry the six lamprey HOX clusters. The assembly also contains several hundred genes that are reproducibly eliminated from somatic cells during early development in lamprey. Comparative analyses show that gnathostome (mouse) homologs of these genes are frequently marked by polycomb repressive complexes (PRCs) in embryonic stem cells, suggesting overlaps in the regulatory logic of somatic DNA elimination and bivalent states that are regulated by early embryonic PRCs. This new assembly will enhance diverse studies that are informed by lampreys’ unique biology and evolutionary/comparative perspective.

Read more
September 22, 2019

Sequence analysis of European maize inbred line F2 provides new insights into molecular and chromosomal characteristics of presence/absence variants.

Maize is well known for its exceptional structural diversity, including copy number variants (CNVs) and presence/absence variants (PAVs), and there is growing evidence for the role of structural variation in maize adaptation. While PAVs have been described in this important crop species, they have been only scarcely characterized at the sequence level and the extent of presence/absence variation and relative chromosomal landscape of inbred-specific regions remain to be elucidated.De novo genome sequencing of the French F2 maize inbred line revealed 10,044 novel genomic regions larger than 1 kb, making up 88 Mb of DNA, that are present in F2 but not in B73 (PAV). This set of maize PAV sequences allowed us to annotate PAV content and to analyze sequence breakpoints. Using PAV genotyping on a collection of 25 temperate lines, we also analyzed Linkage Disequilibrium in PAVs and flanking regions, and PAV frequencies within maize genetic groups.We highlight the possible role of MMEJ-type double strand break repair in maize PAV formation and discover 395 new genes with transcriptional support. Pattern of linkage disequilibrium within PAVs strikingly differs from this of flanking regions and is in accordance with the intuition that PAVs may recombine less than other genomic regions. We show that most PAVs are ancient, while some are found only in European Flint material, thus pinpointing structural features that may be at the origin of adaptive traits involved in the success of this material. Characterization of such PAVs will provide useful material for further association genetic studies in European and temperate maize.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.