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A Histophilus somni isolate from a clinically healthy, fall-placed calf was obtained upon arrival to a commercial feedlot. Fall-placed calves are commonly viewed to be at high risk for the development of bovine respiratory disease. The isolate was phenotyped for antimicrobial susceptibility and sequenced to obtain a complete, circular, genome assembly., Competing Interests: The authors declare no conflict of interest.

Introduction: Bovine respiratory disease (BRD) is one of the most important animal health problems in the beef industry. While bacterial culture and antimicrobial susceptibility testing have been used for diagnostic testing, the common practice of examining one isolate per species does not fully reflect the bacterial population in the sample. In contrast, a recent study with metagenomic sequencing of nasal swabs from feedlot cattle is promising in terms of bacterial pathogen identification and detection of antimicrobial resistance genes (ARGs). However, the sensitivity of metagenomic sequencing was impeded by the high proportion of host biomass in the nasal swab samples., Methods: This pilot study employed a non-selective bacterial enrichment step before nucleic acid extraction to increase the relative proportion of bacterial DNA for sequencing., Results: Non-selective bacterial enrichment increased the proportion of bacteria relative to host sequence data, allowing increased detection of BRD pathogens compared with unenriched samples. This process also allowed for enhanced detection of ARGs with species-level resolution, including detection of ARGs for bacterial species of interest that were not targeted for culture and susceptibility testing. The long-read sequencing approach enabled ARG detection on individual bacterial reads without the need for assembly. Metagenomics following non-selective bacterial enrichment resulted in substantial agreement for four of six comparisons with culture for respiratory bacteria and substantial or better correlation with qPCR. Comparison between isolate susceptibility results and detection of ARGs was best for macrolide ARGs in Mannheimia haemolytica reads but was also substantial for sulfonamide ARGs within M. haemolytica and Pasteurella multocida reads and tetracycline ARGs in Histophilus somni reads., Discussion: By increasing the proportion of bacterial DNA relative to host DNA through non-selective enrichment, we demonstrated a corresponding increase in the proportion of sequencing data identifying BRD-associated pathogens and ARGs in deep nasopharyngeal swabs from feedlot cattle using long-read metagenomic sequencing. This method shows promise as a detection strategy for BRD pathogens and ARGs and strikes a balance between processing time, input costs, and generation of on-target data. This approach could serve as a valuable tool to inform antimicrobial management for BRD and support antimicrobial stewardship., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Herman, Lacoste, Freeman, Otto, McCarthy, Links, Stothard and Waldner.)

Birth mode has been implicated as a major factor influencing neonatal gut microbiome development, and it has been assumed that lack of exposure to the maternal vaginal microbiome is responsible for gut dysbiosis among caesarean-delivered infants. Consequently, practices to correct dysbiotic gut microbiomes, such as vaginal seeding, have arisen while the effect of the maternal vaginal microbiome on that of the infant gut remains unknown. We conducted a longitudinal, prospective cohort study of 621 Canadian pregnant women and their newborn infants and collected pre-delivery maternal vaginal swabs and infant stool samples at 10-days and 3-months of life. Using cpn 60-based amplicon sequencing, we defined vaginal and stool microbiome profiles and evaluated the effect of maternal vaginal microbiome composition and various clinical variables on the development of the infant stool microbiome. Infant stool microbiomes showed significant differences in composition by delivery mode at 10-days postpartum; however, this effect could not be explained by maternal vaginal microbiome composition and was vastly reduced by 3 months. Vaginal microbiome clusters were distributed across infant stool clusters in proportion to their frequency in the overall maternal population, indicating independence of the two communities. Intrapartum antibiotic administration was identified as a confounder of infant stool microbiome differences and was associated with lower abundances of Escherichia coli , Bacteroides vulgatus , Bifidobacterium longum and Parabacteroides distasonis . Our findings demonstrate that maternal vaginal microbiome composition at delivery does not affect infant stool microbiome composition and development, suggesting that practices to amend infant stool microbiome composition focus factors other than maternal vaginal microbes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dos Santos, Pakzad, Albert, Elwood, Grabowska, Links, Hutcheon, Maan, Manges, Dumonceaux, Hodgson, Lyons, Mitchell-Foster, Gantt, Joseph, Van Schalkwyk, Hill and Money.)

Background: Bovine respiratory disease (BRD) is an important cause of morbidity and mortality and is responsible for most of the injectable antimicrobial use in the feedlot industry. Traditional bacterial culture can be used to diagnose BRD by confirming the presence of causative pathogens and to support antimicrobial selection. However, given that bacterial culture takes up to a week and early intervention is critical for treatment success, culture has limited utility for informing rapid therapeutic decision-making. In contrast, metagenomic sequencing has the potential to quickly resolve all nucleic acid in a sample, including pathogen biomarkers and antimicrobial resistance genes. In particular, third-generation Oxford Nanopore Technology sequencing platforms provide long reads and access to raw sequencing data in real-time as it is produced, thereby reducing the time from sample collection to diagnostic answer. The purpose of this study was to compare the performance of nanopore metagenomic sequencing to traditional culture and sensitivity methods as applied to nasopharyngeal samples from segregated groups of chronically ill feedlot cattle, previously treated with antimicrobials for nonresponsive pneumonia or lameness., Results: BRD pathogens were isolated from most samples and a variety of different resistance profiles were observed across isolates. The sequencing data indicated the samples were dominated by Moraxella bovoculi, Mannheimia haemolytica, Mycoplasma dispar, and Pasteurella multocida, and included a wide range of antimicrobial resistance genes (ARGs), encoding resistance for up to seven classes of antimicrobials. Genes conferring resistance to beta-lactams were the most commonly detected, while the tetH gene was detected in the most samples overall. Metagenomic sequencing detected the BRD pathogens of interest more often than did culture, but there was limited concordance between phenotypic resistance to antimicrobials and the presence of relevant ARGs., Conclusions: Metagenomic sequencing can reduce the time from sampling to results, detect pathogens missed by bacterial culture, and identify genetically encoded determinants of resistance. Increasing sequencing coverage of target organisms will be an essential component of improving the reliability of this technology, such that it can be better used for the surveillance of pathogens of interest, genetic determinants of resistance, and to inform diagnostic decisions., (© 2022. The Author(s).)

Background: The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with "universal" PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics., Methods: We present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir., Results: The CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health., Conclusions: cpn60 -based hybridization enriched for taxonomically informative DNA sequences from complex mixtures. In synthetic and natural microbial ecosystems, CaptureSeq provided sequences from prokaryotes and eukaryotes simultaneously, with quantitatively reliable read abundances. CaptureSeq provides an alternative to PCR amplification of taxonomic markers with deep community coverage while minimizing amplification biases.

Modifying the rhizosphere microbiome through targeted plant breeding is key to harnessing positive plant-microbial interrelationships in cropping agroecosystems. Here, we examine the composition of rhizosphere bacterial communities of diverse Brassica napus genotypes to identify: (1) taxa that preferentially associate with genotypes, (2) core bacterial microbiota associated with B. napus , (3) heritable alpha diversity measures at flowering and whole growing season, and (4) correlation between microbial and plant genetic distance among canola genotypes at different growth stages. Our aim is to identify and describe signature microbiota with potential positive benefits that could be integrated in B. napus breeding and management strategies. Rhizosphere soils of 16 diverse genotypes sampled weekly over a 10-week period at single location as well as at three time points at two additional locations were analyzed using 16S rRNA gene amplicon sequencing. The B. napus rhizosphere microbiome was characterized by diverse bacterial communities with 32 named bacterial phyla. The most abundant phyla were Proteobacteria, Actinobacteria, and Acidobacteria. Overall microbial and plant genetic distances were highly correlated ( R = 0.65). Alpha diversity heritability estimates were between 0.16 and 0.41 when evaluated across growth stage and between 0.24 and 0.59 at flowering. Compared with a reference B. napus genotype, a total of 81 genera were significantly more abundant and 71 were significantly less abundant in at least one B. napus genotype out of the total 558 bacterial genera. Most differentially abundant genera were Proteobacteria and Actinobacteria followed by Bacteroidetes and Firmicutes. Here, we also show that B. napus genotypes select an overall core bacterial microbiome with growth-stage-related patterns as to how taxa joined the core membership. In addition, we report that sets of B. napus core taxa were consistent across our three sites and 2 years. Both differential abundance and core analysis implicate numerous bacteria that have been reported to have beneficial effects on plant growth including disease suppression, antifungal properties, and plant growth promotion. Using a multi-site year, temporally intensive field sampling approach, we showed that small plant genetic differences cause predictable changes in canola microbiome and are potential target for direct and indirect selection within breeding programs., (Copyright © 2020 Taye, Helgason, Bell, Norris, Vail, Robinson, Parkin, Arcand, Mamet, Links, Dowhy, Siciliano and Lamb.)

Background: Antimicrobial resistance (AMR) is a major threat to global public health because it makes standard treatments ineffective and contributes to the spread of infections. It is important to understand AMR's biological mechanisms for the development of new drugs and more rapid and accurate clinical diagnostics. The increasing availability of whole-genome SNP (single nucleotide polymorphism) information, obtained from whole-genome sequence data, along with AMR profiles provides an opportunity to use feature selection in machine learning to find AMR-associated mutations. This work describes the use of a supervised feature selection approach using deep neural networks to detect AMR-associated genetic factors from whole-genome SNP data., Results: The proposed method, DNP-AAP (deep neural pursuit - average activation potential), was tested on a Neisseria gonorrhoeae dataset with paired whole-genome sequence data and resistance profiles to five commonly used antibiotics including penicillin, tetracycline, azithromycin, ciprofloxacin, and cefixime. The results show that DNP-AAP can effectively identify known AMR-associated genes in N. gonorrhoeae, and also provide a list of candidate genomic features (SNPs) that might lead to the discovery of novel AMR determinants. Logistic regression classifiers were built with the identified SNPs and the prediction AUCs (area under the curve) for penicillin, tetracycline, azithromycin, ciprofloxacin, and cefixime were 0.974, 0.969, 0.949, 0.994, and 0.976, respectively., Conclusions: DNP-AAP can effectively identify known AMR-associated genes in N. gonorrhoeae. It also provides a list of candidate genes and intergenic regions that might lead to novel AMR factor discovery. More generally, DNP-AAP can be applied to AMR analysis of any bacterial species with genomic variants and phenotype data. It can serve as a useful screening tool for microbiologists to generate genetic candidates for further lab experiments.

We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes.

Microorganisms indigenous to an oil reservoir were grown in media containing either sucrose or proteins in four steel vessels under anoxic conditions at 30°C and 8.3MPa for 30days, to enrich biosurfactant producers. Fermentation of substrate was possible in the protein-containing medium and either fermentation or respiration through reduction of sulfate occurred in the sucrose-containing medium. Growth of microorganisms led to 3.4-5.4-fold surface tension reduction indicating production of biosurfactants in amounts sufficient for enhancement of gas-driven oil recovery. Analysis of sequenced cpn60 amplicons showed that Pseudomonas sp. highly similar to biosurfactant producing P. fluorescens and to Pseudomonas sp. strain TKP predominated, and a bacterium highly similar to biosurfactant producing Bacillus mojavensis was present in vessels. Analysis of 16S rDNA amplicons allowed only genus-level identification of these bacteria. Thus, cpn60-amplicon analysis was a more relevant tool for identification of putative biosurfactant producers than 16S rDNA-amplicon analysis., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

Background: The protist Plasmodiophora brassicae is a soil-borne pathogen of cruciferous species and the causal agent of clubroot disease of Brassicas including agriculturally important crops such as canola/rapeseed (Brassica napus). P. brassicae has remained an enigmatic plant pathogen and is a rare example of an obligate biotroph that resides entirely inside the host plant cell. The pathogen is the cause of severe yield losses and can render infested fields unsuitable for Brassica crop growth due to the persistence of resting spores in the soil for up to 20 years., Results: To provide insight into the biology of the pathogen and its interaction with its primary host B. napus, we produced a draft genome of P. brassicae pathotypes 3 and 6 (Pb3 and Pb6) that differ in their host range. Pb3 is highly virulent on B. napus (but also infects other Brassica species) while Pb6 infects only vegetable Brassica crops. Both the Pb3 and Pb6 genomes are highly compact, each with a total size of 24.2 Mb, and contain less than 2 % repetitive DNA. Clustering of genome-wide single nucleotide polymorphisms (SNP) of Pb3, Pb6 and three additional re-sequenced pathotypes (Pb2, Pb5 and Pb8) shows a high degree of correlation of cluster grouping with host range. The Pb3 genome features significant reduction of intergenic space with multiple examples of overlapping untranslated regions (UTRs). Dependency on the host for essential nutrients is evident from the loss of genes for the biosynthesis of thiamine and some amino acids and the presence of a wide range of transport proteins, including some unique to P. brassicae. The annotated genes of Pb3 include those with a potential role in the regulation of the plant growth hormones cytokinin and auxin. The expression profile of Pb3 genes, including putative effectors, during infection and their potential role in manipulation of host defence is discussed., Conclusion: The P. brassicae genome sequence reveals a compact genome, a dependency of the pathogen on its host for some essential nutrients and a potential role in the regulation of host plant cytokinin and auxin. Genome annotation supported by RNA sequencing reveals significant reduction in intergenic space which, in addition to low repeat content, has likely contributed to the P. brassicae compact genome.

Five avirulence genes from Leptosphaeria maculans, the causal agent of blackleg of canola (Brassica napus), have been identified previously through map-based cloning. In this study, a comparative genomic approach was used to clone the previously mapped AvrLm2. Given the lack of a presence-absence gene polymorphism coincident with the AvrLm2 phenotype, 36 L. maculans isolates were resequenced and analysed for single-nucleotide polymorphisms (SNPs) in predicted small secreted protein-encoding genes present within the map interval. Three SNPs coincident with the AvrLm2 phenotype were identified within LmCys1, previously identified as a putative effector-coding gene. Complementation of a virulent isolate with LmCys1, as the candidate AvrLm2 allele, restored the avirulent phenotype on Rlm2-containing B. napus lines. AvrLm2 encodes a small cysteine-rich protein with low similarity to other proteins in the public databases. Unlike other avirulence genes, AvrLm2 resides in a small GC island within an AT-rich isochore of the genome, and was never found to be deleted completely in virulent isolates., (© 2014 BSPP AND JOHN WILEY & SONS LTD.)

The vaginal microbiota is important in women's reproductive and overall health. However, the relationships between the structure, function and dynamics of this complex microbial community and health outcomes remain elusive. The objective of this study was to determine the phylogenetic range and abundance of prokaryotes in the vaginal microbiota of healthy, non-pregnant, ethnically diverse, reproductive-aged Canadian women. Socio-demographic, behavioural and clinical data were collected and vaginal swabs were analyzed from 310 women. Detailed profiles of their vaginal microbiomes were generated by pyrosequencing of the chaperonin-60 universal target. Six community state types (CST) were delineated by hierarchical clustering, including three Lactobacillus-dominated CST (L. crispatus, L. iners, L. jensenii), two Gardnerella-dominated (subgroups A and C) and an "intermediate" CST which included a small number of women with microbiomes dominated by seven other species or with no dominant species but minority populations of Streptococcus, Staphylococcus, Peptoniphilus, E. coli and various Proteobacteria in co-dominant communities. The striking correspondence between Nugent score and deep sequencing CST continues to reinforce the basic premise provided by the simpler Gram stain method, while additional analyses reveal detailed cpn60-based phylogeny and estimated abundance in microbial communities from vaginal samples. Ethnicity was the only demographic or clinical characteristic predicting CST, with differences in Asian and White women (p = 0.05). In conclusion, this study confirms previous work describing four cpn60-based subgroups of Gardnerella, revealing previously undescribed CST. The data describe the range of bacterial communities seen in Canadian women presenting with no specific vaginal health concerns, and provides an important baseline for future investigations of clinically important cohorts.

Unbiased identification of organisms by PCR reactions using universal primers followed by DNA sequencing assumes positive amplification. We used six universal loci spanning 48 plant species and quantified the bias at each step of the identification process from end point PCR to next-generation sequencing. End point amplification was significantly different for single loci and between species. Quantitative PCR revealed that Cq threshold for various loci, even within a single DNA extraction, showed 2,000-fold differences in DNA quantity after amplification. Next-generation sequencing (NGS) experiments in nine species showed significant biases towards species and specific loci using adaptor-specific primers. NGS sequencing bias may be predicted to some extent by the Cq values of qPCR amplification.

Background: Many plant-pathogenic fungi have a tendency towards genome size expansion, mostly driven by increasing content of transposable elements (TEs). Through comparative and evolutionary genomics, five members of the Leptosphaeria maculans-Leptosphaeria biglobosa species complex (class Dothideomycetes, order Pleosporales), having different host ranges and pathogenic abilities towards cruciferous plants, were studied to infer the role of TEs on genome shaping, speciation, and on the rise of better adapted pathogens., Results: L. maculans 'brassicae', the most damaging species on oilseed rape, is the only member of the species complex to have a TE-invaded genome (32.5%) compared to the other members genomes (<4%). These TEs had an impact at the structural level by creating large TE-rich regions and are suspected to have been instrumental in chromosomal rearrangements possibly leading to speciation. TEs, associated with species-specific genes involved in disease process, also possibly had an incidence on evolution of pathogenicity by promoting translocations of effector genes to highly dynamic regions and thus tuning the regulation of effector gene expression in planta., Conclusions: Invasion of L. maculans 'brassicae' genome by TEs followed by bursts of TE activity allowed this species to evolve and to better adapt to its host, making this genome species a peculiarity within its own species complex as well as in the Pleosporales lineage.

Pseudomonas sp. BRG100 inhibits the growth of certain agricultural pests and is a potentially useful biopesticide for weeds and plant diseases. We have sequenced the 6.25-Mbp genome of this strain and assembled it into 4 scaffolds. Genome sequence comparisons revealed that this strain may represent a novel species of Pseudomonas., (Copyright © 2014 Dumonceaux et al.)

Background: The vaginal microbial community plays a vital role in maintaining women's health. Understanding the precise bacterial composition is challenging because of the diverse and difficult-to-culture nature of many bacterial constituents, necessitating culture-independent methodology. During a natural menstrual cycle, physiological changes could have an impact on bacterial growth, colonization, and community structure. The objective of this study was to assess the stability of the vaginal microbiome of healthy Canadian women throughout a menstrual cycle by using cpn60-based microbiota analysis. Vaginal swabs from 27 naturally cycling reproductive-age women were collected weekly through a single menstrual cycle. Polymerase chain reaction (PCR) was performed to amplify the universal target region of the cpn60 gene and generate amplicons representative of the microbial community. Amplicons were pyrosequenced, assembled into operational taxonomic units, and analyzed. Samples were also assayed for total 16S rRNA gene content and Gardnerella vaginalis by quantitative PCR and screened for the presence of Mollicutes by using family and genus-specific PCR., Results: Overall, the vaginal microbiome of most women remained relatively stable throughout the menstrual cycle, with little variation in diversity and only modest fluctuations in species richness. Microbiomes between women were more different than were those collected consecutively from individual women. Clustering of microbial profiles revealed the expected groupings dominated by Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii. Interestingly, two additional clusters were dominated by either Bifidobacterium breve or a heterogeneous mixture of nonlactobacilli. Direct G. vaginalis quantification correlated strongly with its pyrosequencing-read abundance, and Mollicutes, including Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum, were detected in most samples., Conclusions: Our cpn60-based investigation of the vaginal microbiome demonstrated that in healthy women most vaginal microbiomes remained stable through their menstrual cycle. Of interest in these findings was the presence of Bifidobacteriales beyond just Gardnerella species. Bifidobacteriales are frequently underrepresented in 16S rRNA gene-based studies, and their detection by cpn60-based investigation suggests that their significance in the vaginal community may be underappreciated.

The Brassicaceae (Cruciferae) family, owing to its remarkable species, genetic, and physiological diversity as well as its significant economic potential, has become a model for polyploidy and evolutionary studies. Utilizing extensive transcriptome pyrosequencing of diverse taxa, we established a resolved phylogeny of a subset of crucifer species. We elucidated the frequency, age, and phylogenetic position of polyploidy and lineage separation events that have marked the evolutionary history of the Brassicaceae. Besides the well-known ancient α (47 million years ago [Mya]) and β (124 Mya) paleopolyploidy events, several species were shown to have undergone a further more recent (∼7 to 12 Mya) round of genome multiplication. We identified eight whole-genome duplications corresponding to at least five independent neo/mesopolyploidy events. Although the Brassicaceae family evolved from other eudicots at the beginning of the Cenozoic era of the Earth (60 Mya), major diversification occurred only during the Neogene period (0 to 23 Mya). Remarkably, the widespread species divergence, major polyploidy, and lineage separation events during Brassicaceae evolution are clustered in time around epoch transitions characterized by prolonged unstable climatic conditions. The synchronized diversification of Brassicaceae species suggests that polyploid events may have conferred higher adaptability and increased tolerance toward the drastically changing global environment, thus facilitating species radiation., (© 2014 American Society of Plant Biologists. All rights reserved.)

Background: Brassica oleracea is a valuable vegetable species that has contributed to human health and nutrition for hundreds of years and comprises multiple distinct cultivar groups with diverse morphological and phytochemical attributes. In addition to this phenotypic wealth, B. oleracea offers unique insights into polyploid evolution, as it results from multiple ancestral polyploidy events and a final Brassiceae-specific triplication event. Further, B. oleracea represents one of the diploid genomes that formed the economically important allopolyploid oilseed, Brassica napus. A deeper understanding of B. oleracea genome architecture provides a foundation for crop improvement strategies throughout the Brassica genus., Results: We generate an assembly representing 75% of the predicted B. oleracea genome using a hybrid Illumina/Roche 454 approach. Two dense genetic maps are generated to anchor almost 92% of the assembled scaffolds to nine pseudo-chromosomes. Over 50,000 genes are annotated and 40% of the genome predicted to be repetitive, thus contributing to the increased genome size of B. oleracea compared to its close relative B. rapa. A snapshot of both the leaf transcriptome and methylome allows comparisons to be made across the triplicated sub-genomes, which resulted from the most recent Brassiceae-specific polyploidy event., Conclusions: Differential expression of the triplicated syntelogs and cytosine methylation levels across the sub-genomes suggest residual marks of the genome dominance that led to the current genome architecture. Although cytosine methylation does not correlate with individual gene dominance, the independent methylation patterns of triplicated copies suggest epigenetic mechanisms play a role in the functional diversification of duplicate genes.

Camelina sativa is an oilseed with desirable agronomic and oil-quality attributes for a viable industrial oil platform crop. Here we generate the first chromosome-scale high-quality reference genome sequence for C. sativa and annotated 89,418 protein-coding genes, representing a whole-genome triplication event relative to the crucifer model Arabidopsis thaliana. C. sativa represents the first crop species to be sequenced from lineage I of the Brassicaceae. The well-preserved hexaploid genome structure of C. sativa surprisingly mirrors those of economically important amphidiploid Brassica crop species from lineage II as well as wheat and cotton. The three genomes of C. sativa show no evidence of fractionation bias and limited expression-level bias, both characteristics commonly associated with polyploid evolution. The highly undifferentiated polyploid genome of C. sativa presents significant consequences for breeding and genetic manipulation of this industrial oil crop.

In order to address the hypothesis that seeds from ecologically and geographically diverse plants harbor characteristic epiphytic microbiota, we characterized the bacterial and fungal microbiota associated with Triticum and Brassica seed surfaces. The total microbial complement was determined by amplification and sequencing of a fragment of chaperonin 60 (cpn60). Specific microorganisms were quantified by qPCR. Bacteria and fungi corresponding to operational taxonomic units (OTU) that were identified in the sequencing study were isolated and their interactions examined. A total of 5477 OTU were observed from seed washes. Neither total epiphytic bacterial load nor community richness/evenness was significantly different between the seed types; 578 OTU were shared among all samples at a variety of abundances. Hierarchical clustering revealed that 203 were significantly different in abundance on Triticum seeds compared with Brassica. Microorganisms isolated from seeds showed 99-100% identity between the cpn60 sequences of the isolates and the OTU sequences from this shared microbiome. Bacterial strains identified as Pantoea agglomerans had antagonistic properties toward one of the fungal isolates (Alternaria sp.), providing a possible explanation for their reciprocal abundances on both Triticum and Brassica seeds. cpn60 enabled the simultaneous profiling of bacterial and fungal microbiota and revealed a core seed-associated microbiota shared between diverse plant genera., (© 2014 AAFC. New Phytologist © 2014 New Phytologist Trust.)

A time-course analysis was conducted of thermophilic anaerobic digestion of dairy manure and wheat distillery thin stillage. Sequencing of chaperonin targets provided a phylogenetic survey of both bacteria and archaea in the digestate, along with an appraisal of the diversity of the reactor microbiome. A total of 1129 bacterial operational taxonomic units (OTU) were detected in the reactors, with OTU related to Clostridium becoming numerically dominant by day 7, and Acetivibrio-related OTU by day 35. Archaeal communities were less diverse, with 19 OTU detected representing both acetoclastic and hydrogenotrophic methanogens. Regardless of input material, the same organisms came to dominate the reactors, reflecting strong selective pressures present in the digesters. Principal coordinate analysis of the microbial communities showed that the bacterial communities clustered based on factors other than input material. Bacterial and archaeal OTU were identified with significant correlations to performance parameters, suggesting important roles in the methane production pathway., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Background: Formation of operational taxonomic units (OTU) is a common approach to data aggregation in microbial ecology studies based on amplification and sequencing of individual gene targets. The de novo assembly of OTU sequences has been recently demonstrated as an alternative to widely used clustering methods, providing robust information from experimental data alone, without any reliance on an external reference database., Results: Here we introduce mPUMA (microbial Profiling Using Metagenomic Assembly, http://mpuma.sourceforge.net), a software package for identification and analysis of protein-coding barcode sequence data. It was developed originally for Cpn60 universal target sequences (also known as GroEL or Hsp60). Using an unattended process that is independent of external reference sequences, mPUMA forms OTUs by DNA sequence assembly and is capable of tracking OTU abundance. mPUMA processes microbial profiles both in terms of the direct DNA sequence as well as in the translated amino acid sequence for protein coding barcodes. By forming OTUs and calculating abundance through an assembly approach, mPUMA is capable of generating inputs for several popular microbiota analysis tools. Using SFF data from sequencing of a synthetic community of Cpn60 sequences derived from the human vaginal microbiome, we demonstrate that mPUMA can faithfully reconstruct all expected OTU sequences and produce compositional profiles consistent with actual community structure., Conclusions: mPUMA enables analysis of microbial communities while empowering the discovery of novel organisms through OTU assembly.

The upper respiratory tract microbiome has an important role in respiratory health. Influenza A is a common viral infection that challenges that health, and a well-recognized sequela is bacterial pneumonia. Given this connection, we sought to characterize the upper respiratory tract microbiota of individuals suffering from the pandemic H1N1 influenza A outbreak of 2009 and determine if microbiome profiles could be correlated with patient characteristics. We determined the microbial profiles of 65 samples from H1N1 patients by cpn60 universal target amplification and sequencing. Profiles were examined at the phylum and nearest neighbor "species" levels using the characteristics of patient gender, age, originating health authority, sample type and designation (STAT/non-STAT). At the phylum level, Actinobacteria-, Firmicutes- and Proteobacteria-dominated microbiomes were observed, with none of the patient characteristics showing significant profile composition differences. At the nearest neighbor "species" level, the upper respiratory tract microbiomes were composed of 13-20 "species" and showed a trend towards increasing diversity with patient age. Interestingly, at an individual level, most patients had one to three organisms dominant in their microbiota. A limited number of discrete microbiome profiles were observed, shared among influenza patients regardless of patient status variables. To assess the validity of analyses derived from sequence read abundance, several bacterial species were quantified by quantitative PCR and compared to the abundance of cpn60 sequence read counts obtained in the study. A strong positive correlation between read abundance and absolute bacterial quantification was observed. This study represents the first examination of the upper respiratory tract microbiome using a target other than the 16S rRNA gene and to our knowledge, the first thorough examination of this microbiome during a viral infection.

Members of the rare microbiome can be important components of complex microbial communities. For example, pet dog ownership is a known risk factor for human campylobacteriosis, and Campylobacter is commonly detected in dog feces by targeted assays. However, these organisms have not been detected by metagenomic methods. The goal of this study was to characterize fecal microbiota from healthy and diarrheic pet dogs using two different levels of molecular detection. PCR amplification and pyrosequencing of the universal cpn60 gene target was used to obtain microbial profiles from each dog. To investigate the relatively rare epsilon-proteobacteria component of the microbiome, a molecular enrichment was carried out using a PCR that first amplified the cpn10-cpn60 region from epsilon-proteobacteria, followed by universal cpn60 target amplification and pyrosequencing. From the non-enriched survey, the major finding was a significantly higher proportion of Bacteroidetes, notably Bacteroides vulgatus, in healthy dogs compared to diarrheic dogs. Epsilon-proteobacteria from the genera Helicobacter and Campylobacter were also detected at a low level in the non-enriched profiles of some dogs. Molecular enrichment increased the proportion of epsilon-proteobacteria sequences detected from each dog, as well as identified novel, presumably rare sequences not seen in the non-enriched profiles. Enriched profiles contained known species of Arcobacter, Campylobacter, Flexispira, and Helicobacter and identified two possibly novel species. These findings add to our understanding of the canine fecal microbiome in general, the epsilon-proteobacteria component specifically, and present a novel modification to traditional metagenomic approaches for study of the rare microbiome.

Barcoding with molecular sequences is widely used to catalogue eukaryotic biodiversity. Studies investigating the community dynamics of microbes have relied heavily on gene-centric metagenomic profiling using two genes (16S rRNA and cpn60) to identify and track Bacteria. While there have been criteria formalized for barcoding of eukaryotes, these criteria have not been used to evaluate gene targets for other domains of life. Using the framework of the International Barcode of Life we evaluated DNA barcodes for Bacteria. Candidates from the 16S rRNA gene and the protein coding cpn60 gene were evaluated. Within complete bacterial genomes in the public domain representing 983 species from 21 phyla, the largest difference between median pairwise inter- and intra-specific distances ("barcode gap") was found from cpn60. Distribution of sequence diversity along the ∼555 bp cpn60 target region was remarkably uniform. The barcode gap of the cpn60 universal target facilitated the faithful de novo assembly of full-length operational taxonomic units from pyrosequencing data from a synthetic microbial community. Analysis supported the recognition of both 16S rRNA and cpn60 as DNA barcodes for Bacteria. The cpn60 universal target was found to have a much larger barcode gap than 16S rRNA suggesting cpn60 as a preferred barcode for Bacteria. A large barcode gap for cpn60 provided a robust target for species-level characterization of data. The assembly of consensus sequences for barcodes was shown to be a reliable method for the identification and tracking of novel microbes in metagenomic studies.

Background: Albugo candida is a biotrophic oomycete that parasitizes various species of Brassicaceae, causing a disease (white blister rust) with remarkable convergence in behaviour to unrelated rusts of basidiomycete fungi., Results: A recent genome analysis of the oomycete Hyaloperonospora arabidopsidis suggests that a reduction in the number of genes encoding secreted pathogenicity proteins, enzymes for assimilation of inorganic nitrogen and sulphur represent a genomic signature for the evolution of obligate biotrophy. Here, we report a draft reference genome of a major crop pathogen Albugo candida (another obligate biotrophic oomycete) with an estimated genome of 45.3 Mb. This is very similar to the genome size of a necrotrophic oomycete Pythium ultimum (43 Mb) but less than half that of H. arabidopsidis (99 Mb). Sequencing of A. candida transcripts from infected host tissue and zoosporangia combined with genome-wide annotation revealed 15,824 predicted genes. Most of the predicted genes lack significant similarity with sequences from other oomycetes. Most intriguingly, A. candida appears to have a much smaller repertoire of pathogenicity-related proteins than H. arabidopsidis including genes that encode RXLR effector proteins, CRINKLER-like genes, and elicitins. Necrosis and Ethylene inducing Peptides were not detected in the genome of A. candida. Putative orthologs of tat-C, a component of the twin arginine translocase system, were identified from multiple oomycete genera along with proteins containing putative tat-secretion signal peptides., Conclusion: Albugo candida has a comparatively small genome amongst oomycetes, retains motility of sporangial inoculum, and harbours a much smaller repertoire of candidate effectors than was recently reported for H. arabidopsidis. This minimal gene repertoire could indicate a lack of expansion, rather than a reduction, in the number of genes that signify the evolution of biotrophy in oomycetes.

We report the annotation and analysis of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. We modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. We used Arabidopsis thaliana as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the number of members of gene families present in the genome may contribute to the remarkable morphological plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops.

Resistance to HIV infection in a cohort of commercial sex workers living in Nairobi, Kenya, is linked to mucosal and antiinflammatory factors that may be influenced by the vaginal microbiota. Since bacterial vaginosis (BV), a polymicrobial dysbiosis characterized by low levels of protective Lactobacillus organisms, is an established risk factor for HIV infection, we investigated whether vaginal microbiology was associated with HIV-exposed seronegative (HESN) or HIV-seropositive (HIV(+)) status in this cohort. A subset of 44 individuals was selected for deep-sequencing analysis based on the chaperonin 60 (cpn60) universal target (UT), including HESN individuals (n = 16), other HIV-seronegative controls (HIV-N, n = 16), and HIV(+) individuals (n = 12). Our findings indicate exceptionally high phylogenetic resolution of the cpn60 UT using reads as short as 200 bp, with 54 species in 29 genera detected in this group. Contrary to our initial hypothesis, few differences between HESN and HIV-N women were observed. Several HIV(+) women had distinct profiles dominated by Escherichia coli. The deep-sequencing phylogenetic profile of the vaginal microbiota corresponds closely to BV(+) and BV(-) diagnoses by microscopy, elucidating BV at the molecular level. A cluster of samples with intermediate abundance of Lactobacillus and dominant Gardnerella was identified, defining a distinct BV phenotype that may represent a transitional stage between BV(+) and BV(-). Several alpha- and betaproteobacteria, including the recently described species Variovorax paradoxus, were found to correlate positively with increased Lactobacillus levels that define the BV(-) ("normal") phenotype. We conclude that cpn60 UT is ideally suited to next-generation sequencing technologies for further investigation of microbial community dynamics and mucosal immunity underlying HIV resistance in this cohort.

D.R. Zeigler determined that the sequence identity of bacterial genomes can be predicted accurately using the sequence identities of a corresponding set of genes that meet certain criteria [32]. This three-gene model for comparing bacterial genome pairs requires the determination of the sequence identities for recN, thdF, and rpoA. This involves the generation of approximately 4.2kb of genomic DNA sequence from each organism to be compared, and also normally requires that oligonucleotide primers be designed for amplification and sequencing based on the sequences of closely related organisms. However, we have developed an analogous mathematical model for predicting the sequence identity of whole genomes based on the sequence identity of the 542-567 base pair chaperonin-60 universal target (cpn60 UT). The cpn60 UT is accessible in nearly all bacterial genomes with a single set of universal primers, and its length is such that it can be completely sequenced in one pair of overlapping sequencing reads via di-deoxy sequencing. These mathematical models were applied to a set of Thermoanaerobacter isolates from a wood chip compost pile and it was shown that both the one-gene cpn60 UT-based model and the three-gene model based on recN, rpoA, and thdF predicted that these isolates could be classified as Thermoanaerobacter thermohydrosulfuricus. Furthermore, it was found that the genomic prediction model using cpn60 UT gave similar results to whole-genome sequence alignments over a broad range of taxa, suggesting that this method may have general utility for screening isolates and predicting their taxonomic affiliations., (Crown Copyright © 2011. Published by Elsevier GmbH. All rights reserved.)

The chaperonin-60 universal target (cpn60 UT) is generated from a set of PCR primers and provides a universally conserved, phylogenetically informative sequence signature for determining the composition of microbial communities by DNA sequencing. Pyrosequencing of cpn60 UT amplicons is emerging as a next-generation tool for providing unprecedented sequencing depth and resolution of microbial communities in individual samples. Owing to the increase in sequencing depth, the dynamic range across which the presence and abundance of individual species can be sampled experimentally also increases, significantly improving our ability to investigate microbial community richness and diversity. The flexible format of the pyrosequencing reaction setup combined with the ability to pool samples through the use of multiplexing IDs makes the generation of microbial profiles based on the cpn60 UT both feasible and cost-effective. We describe here the methods we have developed for determining microbial community profiles by pyrosequencing of cpn60 UT amplicons, from generating amplicons to sequencing and data analysis.

The architecture of the Brassica napus genome is marked by its evolutionary origins. The genome of B. napus was formed from the hybridization of two closely related diploid Brassica species, both of which evolved from an hexaploid ancestor. The extensive whole genome duplication events in its near and distant past result in the allotetraploid genome of B. napus maintaining multiple copies of most genes, which predicts a highly complex and redundant transcriptome that can confound any expression analyses. A stringent assembly of 142,399 B. napus expressed sequence tags allowed the development of a well-differentiated set of reference transcripts, which were used as a foundation to assess the efficacy of available tools for identifying and distinguishing transcripts in B. napus; including microarray hybridization and 3' anchored sequence tag capture. Microarray platforms cannot distinguish transcripts derived from the two progenitors or close homologues, although observed differential expression appeared to be biased towards unique transcripts. The use of 3' capture enhanced the ability to unambiguously identify homologues within the B. napus transcriptome but was limited by tag length. The ability to comprehensively catalogue gene expression in polyploid species could be transformed by the application of cost-efficient next generation sequencing technologies that will capture millions of long sequence tags.

The ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif is a transcriptional regulatory motif identified in members of the ethylene-responsive element binding factor, C2H2, and auxin/indole-3-acetic acid families of transcriptional regulators. Sequence comparison of the core EAR motif sites from these proteins revealed two distinct conservation patterns: LxLxL and DLNxxP. Proteins containing these motifs play key roles in diverse biological functions by negatively regulating genes involved in developmental, hormonal, and stress signaling pathways. Through a genome-wide bioinformatics analysis, we have identified the complete repertoire of the EAR repressome in Arabidopsis (Arabidopsis thaliana) comprising 219 proteins belonging to 21 different transcriptional regulator families. Approximately 72% of these proteins contain a LxLxL type of EAR motif, 22% contain a DLNxxP type of EAR motif, and the remaining 6% have a motif where LxLxL and DLNxxP are overlapping. Published in vitro and in planta investigations support approximately 40% of these proteins functioning as negative regulators of gene expression. Comparative sequence analysis of EAR motif sites and adjoining regions has identified additional preferred residues and potential posttranslational modification sites that may influence the functionality of the EAR motif. Homology searches against protein databases of poplar (Populus trichocarpa), grapevine (Vitis vinifera), rice (Oryza sativa), and sorghum (Sorghum bicolor) revealed that the EAR motif is conserved across these diverse plant species. This genome-wide analysis represents the most extensive survey of EAR motif-containing proteins in Arabidopsis to date and provides a resource enabling investigations into their biological roles and the mechanism of EAR motif-mediated transcriptional regulation.

Background: Functional genomics tools provide researchers with the ability to apply high-throughput techniques to determine the function and interaction of a diverse range of genes. Mutagenized plant populations are one such resource that facilitate gene characterisation. They allow complex physiological responses to be correlated with the expression of single genes in planta, through either reverse genetics where target genes are mutagenized to assay the affect, or through forward genetics where populations of mutant lines are screened to identify those whose phenotype diverges from wild type for a particular trait. One limitation of these types of populations is the prevalence of gene redundancy within plant genomes, which can mask the affect of individual genes. Activation or enhancer populations, which not only provide knock-out but also dominant activation mutations, can facilitate the study of such genes., Results: We have developed a population of almost 50,000 activation tagged A. thaliana lines that have been archived as individual lines to the T3 generation. The population is an excellent tool for both reverse and forward genetic screens and has been used successfully to identify a number of novel mutants. Insertion site sequences have been generated and mapped for 15,507 lines to enable further application of the population, while providing a clear distribution of T-DNA insertions across the genome. The population is being screened for a number of biochemical and developmental phenotypes, provisional data identifying novel alleles and genes controlling steps in proanthocyanidin biosynthesis and trichome development is presented., Conclusion: This publicly available population provides an additional tool for plant researcher's to assist with determining gene function for the many as yet uncharacterised genes annotated within the Arabidopsis genome sequence http://aafc-aac.usask.ca/FST. The presence of enhancer elements on the inserted T-DNA molecule allows both knock-out and dominant activation phenotypes to be identified for traits of interest.

We compared dideoxy sequencing of cloned chaperonin-60 universal target (cpn60 UT) amplicons to pyrosequencing of amplicons derived from vaginal microbial communities. In samples pooled from a number of individuals, the pyrosequencing method produced a data set that included virtually all of the sequences that were found within the clone library and revealed an additional level of taxonomic richness. However, the relative abundances of the sequences were different in the two datasets. These observations were expanded and confirmed by the analysis of paired clone library and pyrosequencing datasets from vaginal swabs taken from four individuals. Both for individuals with a normal vaginal microbiota and for those with bacterial vaginosis, the pyrosequencing method revealed a large number of low-abundance taxa that were missed by the clone library approach. In addition, we showed that the pyrosequencing method generates a reproducible profile of microbial community structure in replicate amplifications from the same community. We also compared the taxonomic composition of a vaginal microbial community determined by pyrosequencing of 16S rRNA amplicons to that obtained using cpn60 universal primers. We found that the profiles generated by the two molecular targets were highly similar, with slight differences in the proportional representation of the taxa detected. However, the number of operational taxonomic units was significantly higher in the cpn60 data set, suggesting that the protein-encoding gene provides improved species resolution over the 16S rRNA target. These observations demonstrate that pyrosequencing of cpn60 UT amplicons provides a robust, reliable method for deep sequencing of microbial communities.

Expressed sequence tag (EST) libraries are a powerful tool for gene discovery in plants with genomes that are not fully sequenced. Adonis aestivalis (Ranunculaceae) is unusual among higher plants in that it accumulates large amounts of the valuable red ketocarotenoid astaxanthin in the flower petals, in addition to other carotenoids, and should therefore be a rich source of transcripts involved in carotenoid biosynthesis. To create a resource for gene discovery and increase our understanding of ketocarotenoid biosynthesis in plants, we sequenced 4189 cloned cDNAs from the floral tissues of A. aestivalis. The EST collection was annotated using sequence homology searches comparing the A. aestivalis ESTs with reference databases. ESTs involved in carotenoid biosynthesis were identified based on sequence homology to published sequences and by functional prediction using InterProScan. This study identified expressed transcripts for most of the known genes involved in the carotenoid pathway. Using an Escherichia coli functional expression system, two candidate transcripts from the A. aestivalis cDNA library were functionally characterized and identified as a novel beta-carotene 3-hydroxylase (CrtH2) and a beta-carotene monooxygenase. The expanded use of this EST collection combined with the E. coli expression system as presented here will greatly facilitate the cloning and characterization of candidate genes involved in carotenoid biosynthesis in plants.

Serial analysis of gene expression (SAGE) is a powerful technique which yields a digital measure of gene expression through the sequencing of libraries of specific mRNA-derived fragments, namely SAGE tags. This chapter introduces the methods and software tools that are available for researchers to analyze gene expression through SAGE analysis. A detailed examination of SAGE analysis in Arabidopsis thaliana using the publicly available analysis tool, SaskSAGE, is provided. The use of this software allows the user to maximize the information gained from SAGE experiments in a model system with a fully sequenced genome.

Background: Wheat is an excellent species to study freezing tolerance and other abiotic stresses. However, the sequence of the wheat genome has not been completely characterized due to its complexity and large size. To circumvent this obstacle and identify genes involved in cold acclimation and associated stresses, a large scale EST sequencing approach was undertaken by the Functional Genomics of Abiotic Stress (FGAS) project., Results: We generated 73,521 quality-filtered ESTs from eleven cDNA libraries constructed from wheat plants exposed to various abiotic stresses and at different developmental stages. In addition, 196,041 ESTs for which tracefiles were available from the National Science Foundation wheat EST sequencing program and DuPont were also quality-filtered and used in the analysis. Clustering of the combined ESTs with d2&#95;cluster and TGICL yielded a few large clusters containing several thousand ESTs that were refractory to routine clustering techniques. To resolve this problem, the sequence proximity and "bridges" were identified by an e-value distance graph to manually break clusters into smaller groups. Assembly of the resolved ESTs generated a 75,488 unique sequence set (31,580 contigs and 43,908 singletons/singlets). Digital expression analyses indicated that the FGAS dataset is enriched in stress-regulated genes compared to the other public datasets. Over 43% of the unique sequence set was annotated and classified into functional categories according to Gene Ontology., Conclusion: We have annotated 29,556 different sequences, an almost 5-fold increase in annotated sequences compared to the available wheat public databases. Digital expression analysis combined with gene annotation helped in the identification of several pathways associated with abiotic stress. The genomic resources and knowledge developed by this project will contribute to a better understanding of the different mechanisms that govern stress tolerance in wheat and other cereals.