Phased Assembly of Neo-Sex Chromosomes Reveals Extensive Y Degeneration and Rapid Genome Evolution in Rumex hastatulus.

Y chromosomes are thought to undergo progressive degeneration due to stepwise loss of recombination and subsequent reduction in selection efficiency. However, the timescales and evolutionary forces driving degeneration remain unclear. To investigate the evolution of sex chromosomes on multiple timescales, we generated a high-quality phased genome assembly of the massive older (<10 MYA) and neo (<200,000 yr) sex chromosomes in the XYY cytotype of the dioecious plant Rumex hastatulus and a hermaphroditic outgroup Rumex salicifolius. Our assemblies, supported by fluorescence in situ hybridization, confirmed that the neo-sex chromosomes were formed by two key events: an X-autosome fusion and a reciprocal translocation between the homologous autosome and the Y chromosome. The enormous sex-linked regions of the X (296 Mb) and two Y chromosomes (503 Mb) both evolved from large repeat-rich genomic regions with low recombination; however, the complete loss of recombination on the Y still led to over 30% gene loss and major rearrangements. In the older sex-linked region, there has been a significant increase in transposable element abundance, even into and near genes. In the neo-sex-linked regions, we observed evidence of extensive rearrangements without gene degeneration and loss. Overall, we inferred significant degeneration during the first 10 million years of Y chromosome evolution but not on very short timescales. Our results indicate that even when sex chromosomes emerge from repetitive regions of already-low recombination, the complete loss of recombination on the Y chromosome still leads to a substantial increase in repetitive element content and gene degeneration.

Pseudomonas syringae coffee blight is associated with the horizontal transfer of plasmid-encoded type III effectors.

The emergence of new pathogens is an ongoing threat to human health and agriculture. While zoonotic spillovers received considerable attention, the emergence of crop diseases is less well studied. Here, we identify genomic factors associated with the emergence of Pseudomonas syringae bacterial blight of coffee. Fifty-three P. syringae strains from diseased Brazilian coffee plants were sequenced. Comparative and evolutionary analyses were used to identify loci associated with coffee blight. Growth and symptomology assays were performed to validate the findings. Coffee isolates clustered in three lineages, including primary phylogroups PG3 and PG4, and secondary phylogroup PG11. Genome-wide association study of the primary PG strains identified 37 loci, including five effectors, most of which were encoded on a plasmid unique to the PG3 and PG4 coffee strains. Evolutionary analyses support the emergence of coffee blight in PG4 when the coffee-associated plasmid and associated effectors derived from a divergent plasmid carried by strains associated with other hosts. This plasmid was only recently transferred into PG3. Natural diversity and CRISPR-Cas9 plasmid curing were used to show that strains with the coffee-associated plasmid grow to higher densities and cause more severe disease symptoms in coffee. This work identifies possible evolutionary mechanisms underlying the emergence of a new lineage of coffee pathogens.

Recombination landscape dimorphism and sex chromosome evolution in the dioecious plant Rumex hastatulus.

There is growing evidence from diverse taxa for sex differences in the genomic landscape of recombination, but the causes and consequences of these differences remain poorly understood. Strong recombination landscape dimorphism between the sexes could have important implications for the dynamics of sex chromosome evolution because low recombination in the heterogametic sex can favour the spread of sexually antagonistic alleles. Here, we present a sex-specific linkage map and revised genome assembly of Rumex hastatulus and provide the first evidence and characterization of sex differences in recombination landscape in a dioecious plant. We present data on significant sex differences in recombination, with regions of very low recombination in males covering over half of the genome. This pattern is evident on both sex chromosomes and autosomes, suggesting that pre-existing differences in recombination may have contributed to sex chromosome formation and divergence. Our analysis of segregation distortion suggests that haploid selection due to pollen competition occurs disproportionately in regions with low male recombination. We hypothesize that sex differences in the recombination landscape have contributed to the formation of a large heteromorphic pair of sex chromosomes in R. hastatulus, but more comparative analyses of recombination will be important to investigate this hypothesis further. This article is part of the theme issue 'Sex determination and sex chromosome evolution in land plants'.

Perturbations of the ZED1 pseudokinase activate plant immunity.

The Pseudomonas syringae acetyltransferase HopZ1a is delivered into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the pseudokinase, ZED1. Here we demonstrate that HopZ1a can acetylate members of a family of 'receptor-like cytoplasmic kinases' (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1-ZED1-PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger ZAR1-dependent immunity in planta, all independently of HopZ1a. A ZED1 mutant that mimics acetylation by HopZ1a also triggers immunity in planta, providing evidence that effector-induced perturbations of ZED1 also activate ZAR1. Overall, our results suggest that interactions between these two RLCK families are promoted by perturbations of structural features that distinguish active from inactive kinase domain conformations. We propose that effector-induced interactions between ZED1/ZRK pseudokinases (RLCK family XII) and PBL kinases (RLCK family VII) provide a sensitive mechanism for detecting perturbations of either kinase family to activate ZAR1-mediated ETI.

Arabidopsis ETHYLENE RESPONSE FACTOR 8 (ERF8) has dual functions in ABA signaling and immunity.

BACKGROUND: ETHYLENE RESPONSE FACTOR (ERF) 8 is a member of one of the largest transcription factor families in plants, the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily. Members of this superfamily have been implicated in a wide variety of processes such as development and environmental stress responses. RESULTS: In this study we demonstrated that ERF8 is involved in both ABA and immune signaling. ERF8 overexpression induced programmed cell death (PCD) in Arabidopsis and Nicotiana benthamiana. This PCD was salicylic acid (SA)-independent, suggesting that ERF8 acts downstream or independent of SA. ERF8-induced PCD was abolished by mutations within the ERF-associated amphiphilic repression (EAR) motif, indicating ERF8 induces cell death through its transcriptional repression activity. Two immunity-related mitogen-activated protein kinases, MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) and MPK11, were identified as ERF8-interacting proteins and directly phosphorylated ERF8 in vitro. Four putative MPK phosphorylation sites were identified in ERF8, one of which (Ser103) was determined to be the predominantly phosphorylated residue in vitro, while mutation of all four putative phosphorylation sites partially suppressed ERF8-induced cell death in N. benthamiana. Genome-wide transcriptomic analysis and pathogen growth assays confirmed a positive role of ERF8 in mediating immunity, as ERF8 knockdown or overexpression lines conferred compromised or enhanced resistance against the hemibiotrophic bacterial pathogen Pseudomonas syringae, respectively. CONCLUSIONS: Together these data reveal that the ABA-inducible transcriptional repressor ERF8 has dual roles in ABA signaling and pathogen defense, and further highlight the complex influence of ABA on plant-microbe interactions.

A Practical Guide to Quantitative Interactor Screening with Next-Generation Sequencing (QIS-Seq).

Yeast two-hybrid screens are a powerful approach to identify protein-protein interactions; however, they are typically limited in the number of interactions identified, and lack quantitative values to ascribe confidence scores to the interactions that are obtained. We have developed a high-throughput, quantitative, yeast two-hybrid screening approach coupled with next-generation sequencing. This strategy allows the identification of interacting proteins that are preferentially associated with a bait of interest, and helps eliminate nonspecific interacting proteins. The method is high-throughput, allowing many more baits to be tested and many more candidate interacting proteins to be identified. Quantitative data allows the interactors to be ascribed confidence scores based on their enrichment with particular baits, and can identify both common and rare interacting proteins.

Expression profiles of miRNAs from bovine mammary glands in response to Streptococcus agalactiae-induced mastitis.

This study aimed to describe the expression profiles of microRNAs (miRNAs) from mammary gland tissues collected from dairy cows with Streptococcus agalactiae-induced mastitis and to identify differentially expressed miRNAs related to mastitis. The mammary glands of Chinese Holstein cows were challenged with Streptococcus agalactiae to induce mastitis. Small RNAs were isolated from the mammary tissues of the test and control groups and then sequenced using the Solexa sequencing technology to construct two small RNA libraries. Potential target genes of these differentially expressed miRNAs were predicted using the RNAhybrid software, and KEGG pathways associated with these genes were analysed. A total of 18 555 913 and 20 847 000 effective reads were obtained from the test and control groups, respectively. In total, 373 known and 399 novel miRNAs were detected in the test group, and 358 known and 232 novel miRNAs were uncovered in the control group. A total of 35 differentially expressed miRNAs were identified in the test group compared to the control group, including 10 up-regulated miRNAs and 25 down-regulated miRNAs. Of these miRNAs, miR-223 exhibited the highest degree of up-regulation with an approximately 3-fold increase in expression, whereas miR-26a exhibited the most decreased expression level (more than 2-fold). The RNAhybrid software predicted 18 801 genes as potential targets of these 35 miRNAs. Furthermore, several immune response and signal transduction pathways, including the RIG-I-like receptor signalling pathway, cytosolic DNA sensing pathway and Notch signal pathway, were enriched in these predicted targets. In summary, this study provided experimental evidence for the mechanism underlying the regulation of bovine mastitis by miRNAs and showed that miRNAs might be involved in signal pathways during S. agalactiae-induced mastitis.

NIN-like protein 8 is a master regulator of nitrate-promoted seed germination in Arabidopsis.

Seeds respond to multiple different environmental stimuli that regulate germination. Nitrate stimulates germination in many plants but how it does so remains unclear. Here we show that the Arabidopsis NIN-like protein 8 (NLP8) is essential for nitrate-promoted seed germination. Seed germination in nlp8 loss-of-function mutants does not respond to nitrate. NLP8 functions even in a nitrate reductase-deficient mutant background, and the requirement for NLP8 is conserved among Arabidopsis accessions. NLP8 reduces abscisic acid levels in a nitrate-dependent manner and directly binds to the promoter of CYP707A2, encoding an abscisic acid catabolic enzyme. Genetic analysis shows that NLP8-mediated promotion of seed germination by nitrate requires CYP707A2. Finally, we show that NLP8 localizes to nuclei and unlike NLP7, does not appear to be activated by nitrate-dependent nuclear retention of NLP7, suggesting that seeds have a unique mechanism for nitrate signalling.

Lung microbiota across age and disease stage in cystic fibrosis.

Understanding the significance of bacterial species that colonize and persist in cystic fibrosis (CF) airways requires a detailed examination of bacterial community structure across a broad range of age and disease stage. We used 16S ribosomal RNA sequencing to characterize the lung microbiota in 269 CF patients spanning a 60 year age range, including 76 pediatric samples from patients of age 4-17, and a broad cross-section of disease status to identify features of bacterial community structure and their relationship to disease stage and age. The CF lung microbiota shows significant inter-individual variability in community structure, composition and diversity. The core microbiota consists of five genera - Streptococcus, Prevotella, Rothia, Veillonella and Actinomyces. CF-associated pathogens such as Pseudomonas, Burkholderia, Stenotrophomonas and Achromobacter are less prevalent than core genera, but have a strong tendency to dominate the bacterial community when present. Community diversity and lung function are greatest in patients less than 10 years of age and lower in older age groups, plateauing at approximately age 25. Lower community diversity correlates with worse lung function in a multivariate regression model. Infection by Pseudomonas correlates with age-associated trends in community diversity and lung function.

Transcriptome microRNA profiling of bovine mammary glands infected with Staphylococcus aureus.

MicroRNAs are small non-coding RNA molecules that are important regulators of gene expression at the post-transcriptional level. miRNAs impact the processes of cell proliferation, differentiation and apoptosis. Thus, the regulation of miRNA expression profiles associated with mastitis will be conducive for its control. In this study, Staphylococcus aureus (S. aureus) was administered to the mammary gland of Chinese Holstein cows to construct a bacteria-type mastitis model. Total RNA was isolated from bovine mammary gland tissue samples from the S. aureus-induced mastitis group and controls. miRNAs were analyzed using Solexa sequencing and bioinformatics processing for the experimental group and control group. Two miRNA libraries were constructed respectively. A total of 370 known bovine miRNAs and 341 novel mi RNAs were detected for the S. aureus and 358 known bovine miRNAs and 232 novel miRNAs for control groups. A total of 77 miRNAs in the S. aureus group showed significant differences compared to the control group. GO (Gene Ontology) analysis showed these target genes were involved in the regulation of cells, binding, etc., while KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that these genes were enriched in endocytosis, and olfactory transduction pathways involved in cancer. These results provide an experimental basis to reveal the cause and regulatory mechanism of mastitis and also suggest the potential of miRNAs to serve as biomarkers for the diagnosis of mastitis in dairy cows.

Sequence diversity and gene expression analyses of expansin-related proteins in the white-rot basidiomycete, Phanerochaete carnosa.

Expansin and expansin-related proteins loosen plant cell wall architectures and are widely distributed in several types of organisms, including plants, fungi and bacteria. Here we describe sequence diversity and unique gene expression profiles of multiple expansin-related proteins identified in the basidiomycete, Phanerochaete carnosa. The protein sequences were homologous to loosenin, an expansin-related protein reported in the basidiomycete, Bjerkandera adusta. We identified homologous sequences of each of those P. carnosa proteins in many basidiomycete species. Twelve P. carnosa loosenin-like proteins (LOOLs) were classified into two subgroups according to sequence homology. Conservation of polysaccharide-binding amino acid residues was stricter in subgroup A. Subgroup A sequences included a conserved 8-9 amino acid insertion in a polysaccharide-binding groove whereas subgroup B contained a 12-18 amino acid insertion next to the binding groove. The P. carnosa genome also encodes the expansin-related protein, DREX1, which adopts a loosenin-like structure but has lower sequence homology to other LOOLs. The gene expression analysis of those proteins showed distinct patterns that were not significantly related to subgroupings. The variation in the protein sequences and gene expression patterns, and wide distribution among the basidiomycota, suggest that the diverse cell wall loosening proteins contribute to effective plant cell wall association and utilization by basidiomycetes.

Metatranscriptome of an anaerobic benzene-degrading, nitrate-reducing enrichment culture reveals involvement of carboxylation in benzene ring activation.

The enzymes involved in the initial steps of anaerobic benzene catabolism are not known. To try to elucidate this critical step, a metatranscriptomic analysis was conducted to compare the genes transcribed during the metabolism of benzene and benzoate by an anaerobic benzene-degrading, nitrate-reducing enrichment culture. RNA was extracted from the mixed culture and sequenced without prior mRNA enrichment, allowing simultaneous examination of the active community composition and the differential gene expression between the two treatments. Ribosomal and mRNA sequences attributed to a member of the family Peptococcaceae from the order Clostridiales were essentially only detected in the benzene-amended culture samples, implicating this group in the initial catabolism of benzene. Genes similar to each of two subunits of a proposed benzene-carboxylating enzyme were transcribed when the culture was amended with benzene. Anaerobic benzoate degradation genes from strict anaerobes were transcribed only when the culture was amended with benzene. Genes for other benzoate catabolic enzymes and for nitrate respiration were transcribed in both samples, with those attributed to an Azoarcus species being most abundant. These findings indicate that the mineralization of benzene starts with its activation by a strict anaerobe belonging to the Peptococcaceae, involving a carboxylation step to form benzoate. These data confirm the previously hypothesized syntrophic association between a benzene-degrading Peptococcaceae strain and a benzoate-degrading denitrifying Azoarcus strain for the complete catabolism of benzene with nitrate as the terminal electron acceptor.

Sequencing and annotation of the Ophiostoma ulmi genome.

BACKGROUND: The ascomycete fungus Ophiostoma ulmi was responsible for the initial pandemic of the massively destructive Dutch elm disease in Europe and North America in early 1910. Dutch elm disease has ravaged the elm tree population globally and is a major threat to the remaining elm population. O. ulmi is also associated with valuable biomaterials applications. It was recently discovered that proteins from O. ulmi can be used for efficient transformation of amylose in the production of bioplastics. RESULTS: We have sequenced the 31.5 Mb genome of O.ulmi using Illumina next generation sequencing. Applying both de novo and comparative genome annotation methods, we predict a total of 8639 gene models. The quality of the predicted genes was validated using a variety of data sources consisting of EST data, mRNA-seq data and orthologs from related fungal species. Sequence-based computational methods were used to identify candidate virulence-related genes. Metabolic pathways were reconstructed and highlight specific enzymes that may play a role in virulence. CONCLUSIONS: This genome sequence will be a useful resource for further research aimed at understanding the molecular mechanisms of pathogenicity by O. ulmi. It will also facilitate the identification of enzymes necessary for industrial biotransformation applications.

Forward chemical genetic screens in Arabidopsis identify genes that influence sensitivity to the phytotoxic compound sulfamethoxazole.

BACKGROUND: The sulfanilamide family comprises a clinically important group of antimicrobial compounds which also display bioactivity in plants. While there is evidence that sulfanilamides inhibit folate biosynthesis in both bacteria and plants, the complete network of plant responses to these compounds remains to be characterized. As such, we initiated two forward genetic screens in Arabidopsis in order to identify mutants that exhibit altered sensitivity to sulfanilamide compounds. These screens were based on the growth phenotype of seedlings germinated in the presence of the compound sulfamethoxazole (Smex). RESULTS: We identified a mutant with reduced sensitivity to Smex, and subsequent mapping indicated that a gene encoding 5-oxoprolinase was responsible for this phenotype. A mutation causing enhanced sensitivity to Smex was mapped to a gene lacking any functional annotation. CONCLUSIONS: The genes identified through our forward genetic screens represent novel mediators of Arabidopsis responses to sulfanilamides and suggest that these responses extend beyond the perturbation of folate biosynthesis.

Analysis of the cystic fibrosis lung microbiota via serial Illumina sequencing of bacterial 16S rRNA hypervariable regions.

The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.

Comparative genomics of the white-rot fungi, Phanerochaete carnosa and P. chrysosporium, to elucidate the genetic basis of the distinct wood types they colonize.

BACKGROUND: Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus, Phanerochaete carnosa, has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by a white-rot fungus, the present study reports the P. carnosa genome sequence and its comparative analysis with the previously reported P. chrysosporium genome. RESULTS: P. carnosa encodes a complete set of lignocellulose-active enzymes. Comparative genomic analysis revealed that P. carnosa is enriched with genes encoding manganese peroxidase, and that the most divergent glycoside hydrolase families were predicted to encode hemicellulases and glycoprotein degrading enzymes. Most remarkably, P. carnosa possesses one of the largest P450 contingents (266 P450s) among the sequenced and annotated wood-rotting basidiomycetes, nearly double that of P. chrysosporium. Along with metabolic pathway modeling, comparative growth studies on model compounds and chemical analyses of decomposed wood components showed greater tolerance of P. carnosa to various substrates including coniferous heartwood. CONCLUSIONS: The P. carnosa genome is enriched with genes that encode P450 monooxygenases that can participate in extractives degradation, and manganese peroxidases involved in lignin degradation. The significant expansion of P450s in P. carnosa, along with differences in carbohydrate- and lignin-degrading enzymes, could be correlated to the utilization of heartwood and sapwood preparations from both coniferous and hardwood species.

Extensive remodeling of the Pseudomonas syringae pv. avellanae type III secretome associated with two independent host shifts onto hazelnut.

BACKGROUND: Hazelnut (Corylus avellana) decline disease in Greece and Italy is caused by the convergent evolution of two distantly related lineages of Pseudomonas syringae pv. avellanae (Pav). We sequenced the genomes of three Pav isolates to determine if their convergent virulence phenotype had a common genetic basis due to either genetic exchange between lineages or parallel evolution. RESULTS: We found little evidence for horizontal transfer (recombination) of genes between Pav lineages, but two large genomic islands (GIs) have been recently acquired by one of the lineages. Evolutionary analyses of the genes encoding type III secreted effectors (T3SEs) that are translocated into host cells and are important for both suppressing and eliciting defense responses show that the two Pav lineages have dramatically different T3SE profiles, with only two shared putatively functional T3SEs. One Pav lineage has undergone unprecedented secretome remodeling, including the acquisition of eleven new T3SEs and the loss or pseudogenization of 15, including five of the six core T3SE families that are present in the other Pav lineage. Molecular dating indicates that divergence within both of the Pav lineages predates their observation in the field. This suggest that both Pav lineages have been cryptically infecting hazelnut trees or wild relatives for many years, and that the emergence of hazelnut decline in the 1970s may have been due to changes in agricultural practice. CONCLUSIONS: These data show that divergent lineages of P. syringae can converge on identical disease etiology on the same host plant using different virulence mechanisms and that dramatic shifts in the arsenal of T3SEs can accompany disease emergence.

Quantitative Interactor Screening with next-generation Sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Pseudomonas syringae type III effector HopZ2.

BACKGROUND: Identification of protein-protein interactions is a fundamental aspect of understanding protein function. A commonly used method for identifying protein interactions is the yeast two-hybrid system. RESULTS: Here we describe the application of next-generation sequencing to yeast two-hybrid interaction screens and develop Quantitative Interactor Screen Sequencing (QIS-Seq). QIS-Seq provides a quantitative measurement of enrichment for each interactor relative to its frequency in the library as well as its general stickiness (non-specific binding). The QIS-Seq approach is scalable and can be used with any yeast two-hybrid screen and with any next-generation sequencing platform. The quantitative nature of QIS-Seq data make it amenable to statistical evaluation, and importantly, facilitates the standardization of experimental design, data collection, and data analysis. We applied QIS-Seq to identify the Arabidopsis thaliana MLO2 protein as a target of the Pseudomonas syringae type III secreted effector protein HopZ2. We validate the interaction between HopZ2 and MLO2 in planta and show that the interaction is required for HopZ2-associated virulence. CONCLUSIONS: We demonstrate that QIS-Seq is a high-throughput quantitative interactor screen and validate MLO2 as an interactor and novel virulence target of the P. syringae type III secreted effector HopZ2.

Semi-automatic in silico gap closure enabled de novo assembly of two Dehalobacter genomes from metagenomic data.

Typically, the assembly and closure of a complete bacterial genome requires substantial additional effort spent in a wet lab for gap resolution and genome polishing. Assembly is further confounded by subspecies polymorphism when starting from metagenome sequence data. In this paper, we describe an in silico gap-resolution strategy that can substantially improve assembly. This strategy resolves assembly gaps in scaffolds using pre-assembled contigs, followed by verification with read mapping. It is capable of resolving assembly gaps caused by repetitive elements and subspecies polymorphisms. Using this strategy, we realized the de novo assembly of the first two Dehalobacter genomes from the metagenomes of two anaerobic mixed microbial cultures capable of reductive dechlorination of chlorinated ethanes and chloroform. Only four additional PCR reactions were required even though the initial assembly with Newbler v. 2.5 produced 101 contigs within 9 scaffolds belonging to two Dehalobacter strains. By applying this strategy to the re-assembly of a recently published genome of Bacteroides, we demonstrate its potential utility for other sequencing projects, both metagenomic and genomic.

Use of low-coverage, large-insert, short-read data for rapid and accurate generation of enhanced-quality draft Pseudomonas genome sequences.

Next-generation genomic technology has both greatly accelerated the pace of genome research as well as increased our reliance on draft genome sequences. While groups such as the Genomics Standards Consortium have made strong efforts to promote genome standards there is a still a general lack of uniformity among published draft genomes, leading to challenges for downstream comparative analyses. This lack of uniformity is a particular problem when using standard draft genomes that frequently have large numbers of low-quality sequencing tracts. Here we present a proposal for an "enhanced-quality draft" genome that identifies at least 95% of the coding sequences, thereby effectively providing a full accounting of the genic component of the genome. Enhanced-quality draft genomes are easily attainable through a combination of small- and large-insert next-generation, paired-end sequencing. We illustrate the generation of an enhanced-quality draft genome by re-sequencing the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola 1448A (Pph 1448A), which has a published, closed genome sequence of 5.93 Mbp. We use a combination of Illumina paired-end and mate-pair sequencing, and surprisingly find that de novo assemblies with 100x paired-end coverage and mate-pair sequencing with as low as low as 2-5x coverage are substantially better than assemblies based on higher coverage. The rapid and low-cost generation of large numbers of enhanced-quality draft genome sequences will be of particular value for microbial diagnostics and biosecurity, which rely on precise discrimination of potentially dangerous clones from closely related benign strains.