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Background: Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species.Results: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans.Conclusions: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae. © 2010 Lévesque et al.; licensee BioMed Central Ltd.

We have investigated the elastic properties of epitaxial MnTe layers using triple axis high resolution x-ray diffraction and reciprocal space mapping. A series CdTe/MnTe superlattices (SLs) grown by molecular beam epitaxy and nearly strain compensated, were deposited on [001] Cd[sub 1 − x]Zn[sub x]Te substrates. In order to obtain the MnTe content of these SLs without an a priori knowledge of the elastic properties of cubic MnTe, annealing experiments were performed to interdiffuse the individual layers into a mixed Cd[sub 1 − x]Mn[sub x] Te alloy layer. For a precise analysis of the data, it was found to be important to determine the in-plane strain of the superlattice layers using reciprocal space maps around symmetric and asymmetric reciprocal lattice points. The value for the Poisson ratio of zinc-blende MnTe was determined to be v = C[sub 11]/2C[sub 12] = 0.77±0.15. [ABSTRACT FROM AUTHOR]

Abstract Background We are developing a cross-species comparison strategy to distinguish between cancer driver- and passenger gene alteration candidates, by utilizing the difference in genomic location of orthologous genes between the human and other mammals. As an initial test of this strategy, we conducted a pilot study with human colorectal cancer (CRC) and its mouse model C57BL/6J ApcMin/+, focusing on human 5q22.2 and 18q21.1-q21.2. Methods We first performed bioinformatics analysis on the evolution of 5q22.2 and 18q21.1-q21.2 regions. Then, we performed exon-targeted sequencing, real time quantitative polymerase chain reaction (qPCR), and real time quantitative reverse transcriptase PCR (qRT-PCR) analyses on a number of genes of both regions with both human and mouse colon tumors. Results These two regions (5q22.2 and 18q21.1-q21.2) are frequently deleted in human CRCs and encode genuine colorectal tumor suppressors APC and SMAD4. They also encode genes such as MCC (mutated in colorectal cancer) with their role in CRC etiology unknown. We have discovered that both regions are evolutionarily unstable, resulting in genes that are clustered in each human region being found scattered at several distinct loci in the genome of many other species. For instance, APC and MCC are within 200 kb apart in human 5q22.2 but are 10 Mb apart in the mouse genome. Importantly, our analyses revealed that, while known CRC driver genes APC and SMAD4 were disrupted in both human colorectal tumors and tumors from ApcMin/+ mice, the questionable MCC gene was disrupted in human tumors but appeared to be intact in mouse tumors. Conclusions These results indicate that MCC may not actually play any causative role in early colorectal tumorigenesis. We also hypothesize that its disruption in human CRCs is likely a mere result of its close proximity to APC in the human genome. Expanding this pilot study to the entire genome may identify more questionable genes like MCC, facilitating the discovery of new CRC driver gene candidates.

Abstract Background Polymerase chain reaction (PCR) is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment. Results We have developed a fully integrated computational PCR primer design pipeline that plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions defined through a rich set of descriptors, such as Ensembl accessions and arbitrary genomic coordinates. Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving our sequencing success rate, which currently exceeds 95% for exons, we have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. We reveal the laboratory protocols and their associated, empirically determined computational parameters, as well as describe the novel computational methods which may benefit others in future primer design research. Conclusion The high-throughput PCR primer design pipeline has been very successful in providing the basis for high-quality directed sequencing results and for minimizing costs associated with labor and reprocessing. The modular architecture of the primer design software has made it possible to readily integrate additional primer critique tests based on iterative feedback from the laboratory. As a result, the primer design software, coupled with the laboratory protocols, serves as a powerful tool for low and high-throughput primer design to enable successful directed sequencing.

The depletion of disruptive variation caused by purifying natural selection (constraint) has been widely used to investigate protein-coding genes underlying human disorders 1-4 , but attempts to assess constraint for non-protein-coding regions have proved more difficult. Here we aggregate, process and release a dataset of 76,156 human genomes from the Genome Aggregation Database (gnomAD)-the largest public open-access human genome allele frequency reference dataset-and use it to build a genomic constraint map for the whole genome (genomic non-coding constraint of haploinsufficient variation (Gnocchi)). We present a refined mutational model that incorporates local sequence context and regional genomic features to detect depletions of variation. As expected, the average constraint for protein-coding sequences is stronger than that for non-coding regions. Within the non-coding genome, constrained regions are enriched for known regulatory elements and variants that are implicated in complex human diseases and traits, facilitating the triangulation of biological annotation, disease association and natural selection to non-coding DNA analysis. More constrained regulatory elements tend to regulate more constrained protein-coding genes, which in turn suggests that non-coding constraint can aid the identification of constrained genes that are as yet unrecognized by current gene constraint metrics. We demonstrate that this genome-wide constraint map improves the identification and interpretation of functional human genetic variation., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Genetic studies in underrepresented populations identify disproportionate numbers of novel associations. However, most genetic studies use genotyping arrays and sequenced reference panels that best capture variation most common in European ancestry populations. To compare data generation strategies best suited for underrepresented populations, we sequenced the whole genomes of 91 individuals to high coverage as part of the Neuropsychiatric Genetics of African Population-Psychosis (NeuroGAP-Psychosis) study with participants from Ethiopia, Kenya, South Africa, and Uganda. We used a downsampling approach to evaluate the quality of two cost-effective data generation strategies, GWAS arrays versus low-coverage sequencing, by calculating the concordance of imputed variants from these technologies with those from deep whole-genome sequencing data. We show that low-coverage sequencing at a depth of ≥4× captures variants of all frequencies more accurately than all commonly used GWAS arrays investigated and at a comparable cost. Lower depths of sequencing (0.5-1×) performed comparably to commonly used low-density GWAS arrays. Low-coverage sequencing is also sensitive to novel variation; 4× sequencing detects 45% of singletons and 95% of common variants identified in high-coverage African whole genomes. Low-coverage sequencing approaches surmount the problems induced by the ascertainment of common genotyping arrays, effectively identify novel variation particularly in underrepresented populations, and present opportunities to enhance variant discovery at a cost similar to traditional approaches., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-function variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes 1 . Here we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence predicted loss-of-function variants in this cohort after filtering for artefacts caused by sequencing and annotation errors. Using an improved model of human mutation rates, we classify human protein-coding genes along a spectrum that represents tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve the power of gene discovery for both common and rare diseases.

Background: Here we present an in-depth characterization of the mechanism of sequencer-induced sample contamination due to the phenomenon of index swapping that impacts Illumina sequencers employing patterned flow cells with Exclusion Amplification (ExAmp) chemistry (HiSeqX, HiSeq4000, and NovaSeq). We also present a remediation method that minimizes the impact of such swaps., Results: Leveraging data collected over a two-year period, we demonstrate the widespread prevalence of index swapping in patterned flow cell data. We calculate mean swap rates across multiple sample preparation methods and sequencer models, demonstrating that different library methods can have vastly different swapping rates and that even non-ExAmp chemistry instruments display trace levels of index swapping. We provide methods for eliminating sample data cross contamination by utilizing non-redundant dual indexing for complete filtering of index swapped reads, and share the sequences for 96 non-combinatorial dual indexes we have validated across various library preparation methods and sequencer models. Finally, using computational methods we provide a greater insight into the mechanism of index swapping., Conclusions: Index swapping in pooled libraries is a prevalent phenomenon that we observe at a rate of 0.2 to 6% in all sequencing runs on HiSeqX, HiSeq 4000/3000, and NovaSeq. Utilizing non-redundant dual indexing allows for the removal (flagging/filtering) of these swapped reads and eliminates swapping induced sample contamination, which is critical for sensitive applications such as RNA-seq, single cell, blood biopsy using circulating tumor DNA, or clinical sequencing.

Photobacterium profundum is a cosmopolitan marine bacterium capable of growth at low temperature and high hydrostatic pressure. Multiple strains of P. profundum have been isolated from different depths of the ocean and display remarkable differences in their physiological responses to pressure. The genome sequence of the deep-sea piezopsychrophilic strain Photobacterium profundum SS9 has provided some clues regarding the genetic features required for growth in the deep sea. The sequenced genome of Photobacterium profundum strain 3TCK, a non-piezophilic strain isolated from a shallow-water environment, is now available and its analysis expands the identification of unique genomic features that correlate to environmental differences and define the Hutchinsonian niche of each strain. These differences range from variations in gene content to specific gene sequences under positive selection. Genome plasticity between Photobacterium bathytypes was investigated when strain 3TCK-specific genes involved in photorepair were introduced to SS9, demonstrating that horizontal gene transfer can provide a mechanism for rapid colonisation of new environments.

Here, we present the draft genome sequence of "Candidatus Nitrosoarchaeum limnia" BG20, an ammonia-oxidizing archaeon enriched in culture from low-salinity sediments of the San Francisco Bay estuary. The genome sequence revealed many similarities to the previously sequenced genome of "Ca. Nitrosoarchaeum limnia" SFB1 (enriched from a nearby site in San Francisco Bay) and is representative of a clade of ammonia-oxidizing archaea (AOA) found in low-salinity habitats worldwide.

Ammonia-oxidizing archaea (AOA) play important roles in nitrogen and carbon cycling in marine and terrestrial ecosystems. Here, we present the draft genome sequence for the ammonia-oxidizing archaeon "Candidatus Nitrosopumilus salaria" BD31, which was enriched in culture from sediments of the San Francisco Bay estuary. The genome sequences revealed many similarities to the genome of Nitrosopumilus maritimus.

Caminibacter mediatlanticus strain TB-2(T) [1], is a thermophilic, anaerobic, chemolithoautotrophic bacterium, isolated from the walls of an active deep-sea hydrothermal vent chimney on the Mid-Atlantic Ridge and the type strain of the species. C. mediatlanticus is a Gram-negative member of the Epsilonproteobacteria (order Nautiliales) that grows chemolithoautotrophically with H(2) as the energy source and CO(2) as the carbon source. Nitrate or sulfur is used as the terminal electron acceptor, with resulting production of ammonium and hydrogen sulfide, respectively. In view of the widespread distribution, importance and physiological characteristics of thermophilic Epsilonproteobacteria in deep-sea geothermal environments, it is likely that these organisms provide a relevant contribution to both primary productivity and the biogeochemical cycling of carbon, nitrogen and sulfur at hydrothermal vents. Here we report the main features of the genome of C. mediatlanticus strain TB-2(T).

Methylophaga thiooxydans is a mesophilic, obligately halophilic bacterium that is capable of methylotrophic growth on a range of one-carbon compounds as well as chemolithoheterotrophic growth at the expense of thiosulfate. Here we present the draft genome sequence of Methylophaga thiooxydans DMS010 (DSM 22068(T), VKM B2586(T)), the type strain of the species, which has allowed prediction of the genes involved in one-carbon metabolism, nitrogen metabolism, and other aspects of central metabolism.

Bacteria are the primary food source of choanoflagellates, the closest known relatives of animals. Studying signaling interactions between the Gram-negative Bacteroidetes bacterium Algoriphagus sp. PR1 and its predator, the choanoflagellate Salpingoeca rosetta, provides a promising avenue for testing hypotheses regarding the involvement of bacteria in animal evolution. Here we announce the complete genome sequence of Algoriphagus sp. PR1 and initial findings from its annotation.

Mariprofundus ferrooxydans PV-1 has provided the first genome of the recently discovered Zetaproteobacteria subdivision. Genome analysis reveals a complete TCA cycle, the ability to fix CO(2), carbon-storage proteins and a sugar phosphotransferase system (PTS). The latter could facilitate the transport of carbohydrates across the cell membrane and possibly aid in stalk formation, a matrix composed of exopolymers and/or exopolysaccharides, which is used to store oxidized iron minerals outside the cell. Two-component signal transduction system genes, including histidine kinases, GGDEF domain genes, and response regulators containing CheY-like receivers, are abundant and widely distributed across the genome. Most of these are located in close proximity to genes required for cell division, phosphate uptake and transport, exopolymer and heavy metal secretion, flagellar biosynthesis and pilus assembly suggesting that these functions are highly regulated. Similar to many other motile, microaerophilic bacteria, genes encoding aerotaxis as well as antioxidant functionality (e.g., superoxide dismutases and peroxidases) are predicted to sense and respond to oxygen gradients, as would be required to maintain cellular redox balance in the specialized habitat where M. ferrooxydans resides. Comparative genomics with other Fe(II) oxidizing bacteria residing in freshwater and marine environments revealed similar content, synteny, and amino acid similarity of coding sequences potentially involved in Fe(II) oxidation, signal transduction and response regulation, oxygen sensation and detoxification, and heavy metal resistance. This study has provided novel insights into the molecular nature of Zetaproteobacteria.

Gene duplication may be an important mechanism for the evolution of new functions and for the adaptive modulation of gene expression via dosage effects. Here, we analyzed the fate of gene duplicates for two strains of a novel group of cyanobacteria (genus Acaryochloris) that produces the far-red light absorbing chlorophyll d as its main photosynthetic pigment. The genomes of both strains contain an unusually high number of gene duplicates for bacteria. As has been observed for eukaryotic genomes, we find that the demography of gene duplicates can be well modeled by a birth-death process. Most duplicated Acaryochloris genes are of comparatively recent origin, are strain-specific, and tend to be located on different genetic elements. Analyses of selection on duplicates of different divergence classes suggest that a minority of paralogs exhibit near neutral evolutionary dynamics immediately following duplication but that most duplicate pairs (including those which have been retained for long periods) are under strong purifying selection against amino acid change. The likelihood of duplicate retention varied among gene functional classes, and the pronounced differences between strains in the pool of retained recent duplicates likely reflects differences in the nutrient status and other characteristics of their respective environments. We conclude that most duplicates are quickly purged from Acaryochloris genomes and that those which are retained likely make important contributions to organism ecology by conferring fitness benefits via gene dosage effects. The mechanism of enhanced duplication may involve homologous recombination between genetic elements mediated by paralogous copies of recA.

Janibacter sp. strain HTCC2649 is a novel marine member of the Actinobacteria, family Intrasporangiaceae, and is closely related to Janibacter melonis CM2104(T) and Knoellia sinensis HKI 0119(T). The organism was isolated from a sample collected at Hydrostation S south of Bermuda by using high-throughput culturing techniques. Here we present the genome sequence of Janibacter sp. strain HTCC2649.

The understanding of marine microbial ecology and metabolism has been hampered by the paucity of sequenced reference genomes. To this end, we report the sequencing of 137 diverse marine isolates collected from around the world. We analysed these sequences, along with previously published marine prokaryotic genomes, in the context of marine metagenomic data, to gain insights into the ecology of the surface ocean prokaryotic picoplankton (0.1-3.0 μm size range). The results suggest that the sequenced genomes define two microbial groups: one composed of only a few taxa that are nearly always abundant in picoplanktonic communities, and the other consisting of many microbial taxa that are rarely abundant. The genomic content of the second group suggests that these microbes are capable of slow growth and survival in energy-limited environments, and rapid growth in energy-rich environments. By contrast, the abundant and cosmopolitan picoplanktonic prokaryotes for which there is genomic representation have smaller genomes, are probably capable of only slow growth and seem to be relatively unable to sense or rapidly acclimate to energy-rich conditions. Their genomic features also lead us to propose that one method used to avoid predation by viruses and/or bacterivores is by means of slow growth and the maintenance of low biomass.

Pelagibaca bermudensis HTCC2601(T) and Maritimibacter alkaliphilus HTCC2654(T) represent two marine genera in the globally significant Roseobacter clade of the Alphaproteobacteria. Here, we present the genome sequences of these organisms, isolated from the Sargasso Sea using dilution-to-extinction culturing, which offer insight into the genetic basis for the metabolic and ecological diversity of this important group.

Fulvimarina pelagi is a Mn(II)-oxidizing marine heterotrophic bacterium in the order Rhizobiales. Here we announce the draft genome sequence of F. pelagi HTCC2506(T), which was isolated from the Sargasso Sea by using dilution-to-extinction culturing. The genome sequence contained a xanthorhodopsin gene as well as a photosynthetic gene cluster, which suggests the coexistence of two different phototrophic mechanisms in a single microorganism.

Strain HTCC2143 was isolated from Oregon Coast surface waters using dilution-to-extinction culturing. Here we present the genome of strain HTCC2143 from the BD1-7 clade of the oligotrophic marine Gammaproteobacteria group. The genome of HTCC2143 contains genes for carotenoid biosynthesis and proteorhodopsin and for proteins that have potential biotechnological significance: epoxide hydrolases, Baeyer-Villiger monooxygenases, and polyketide synthases.

Here we announce the complete genome sequence of Croceibacter atlanticus HTCC2559(T), which was isolated by high-throughput dilution-to-extinction culturing from the Bermuda Atlantic Time Series station in the Western Sargasso Sea. Strain HTCC2559(T) contained genes for carotenoid biosynthesis, flavonoid biosynthesis, and several macromolecule-degrading enzymes. The genome confirmed physiological observations of cultivated Croceibacter atlanticus strain HTCC2559(T), which identified it as an obligate chemoheterotroph.

HTCC5015 is a novel, highly divergent marine member of the Gammaproteobacteria, currently without a cultured representative with greater than 89% 16S rRNA gene identity to itself. The organism was isolated from water collected from Hydrostation S south of Bermuda using high-throughput dilution-to-extinction culturing techniques. Here we present the genome sequence of the unique Gammaproteobacterium strain HTCC5015.

Organisms in the OM60/NOR5 clade of the Gammaproteobacteria are ubiquitous in the world's oceans and can make up as much as 11% of bacterial cells in certain areas. Isolated from coastal Oregon water, Gammaproteobacteria HTCC2148 and HTCC2080 are two members of this important clade. Here we present the genome sequences of the OM60 Gammaproteobacteria HTCC2148 and HTCC2080.

The human microbiome refers to the community of microorganisms, including prokaryotes, viruses, and microbial eukaryotes, that populate the human body. The National Institutes of Health launched an initiative that focuses on describing the diversity of microbial species that are associated with health and disease. The first phase of this initiative includes the sequencing of hundreds of microbial reference genomes, coupled to metagenomic sequencing from multiple body sites. Here we present results from an initial reference genome sequencing of 178 microbial genomes. From 547,968 predicted polypeptides that correspond to the gene complement of these strains, previously unidentified ("novel") polypeptides that had both unmasked sequence length greater than 100 amino acids and no BLASTP match to any nonreference entry in the nonredundant subset were defined. This analysis resulted in a set of 30,867 polypeptides, of which 29,987 (approximately 97%) were unique. In addition, this set of microbial genomes allows for approximately 40% of random sequences from the microbiome of the gastrointestinal tract to be associated with organisms based on the match criteria used. Insights into pan-genome analysis suggest that we are still far from saturating microbial species genetic data sets. In addition, the associated metrics and standards used by our group for quality assurance are presented.

Robiginitalea biformata HTCC2501, isolated from the Sargasso Sea by dilution-to-extinction culturing, has been known as an aerobic chemoheterotroph with carotenoid pigments and dimorphic growth phases. Here, we announce the complete sequence of the R. biformata HTCC2501 genome, which contains genes for carotenoid biosynthesis and several macromolecule-degrading enzymes.

Many marine bacteria have evolved to grow optimally at either high (copiotrophic) or low (oligotrophic) nutrient concentrations, enabling different species to colonize distinct trophic habitats in the oceans. Here, we compare the genome sequences of two bacteria, Photobacterium angustum S14 and Sphingopyxis alaskensis RB2256, that serve as useful model organisms for copiotrophic and oligotrophic modes of life and specifically relate the genomic features to trophic strategy for these organisms and define their molecular mechanisms of adaptation. We developed a model for predicting trophic lifestyle from genome sequence data and tested >400,000 proteins representing >500 million nucleotides of sequence data from 126 genome sequences with metagenome data of whole environmental samples. When applied to available oceanic metagenome data (e.g., the Global Ocean Survey data) the model demonstrated that oligotrophs, and not the more readily isolatable copiotrophs, dominate the ocean's free-living microbial populations. Using our model, it is now possible to define the types of bacteria that specific ocean niches are capable of sustaining.

Erythrobacter litoralis has been known as a bacteriochlorophyll a-containing, aerobic, anoxygenic, phototrophic bacterium. Here we announce the complete genome sequence of E. litoralis HTCC2594, which is devoid of phototrophic potential. E. litoralis HTCC2594, isolated by dilution-to-extinction culturing from seawater, could not carry out aerobic anoxygenic phototrophy and lacked genes for bacteriochlorophyll a biosynthesis and photosynthetic reaction center proteins.

Alteromonas macleodii is a common marine heterotrophic gamma-proteobacterium. Isolates from this microbe cluster by molecular analysis into two major genotypic groups or ecotypes, one found in temperate latitudes in the upper water column and another that is for the most part found in the deep water column of the Mediterranean. Here, we describe the genome of one strain of the 'deep ecotype' (AltDE) isolated from 1000 m in the Eastern Mediterranean and compare this genome with that of the type strain ATCC 27126, a representative of the global 'surface' ecotype. The genomes are substantially different with DNA sequence similarity values that are borderline for microbes belonging to the same species, and a large differential gene content, mainly found in islands larger than 20 kbp, that also recruit poorly to the Global Ocean Sampling project (GOS). These genomic differences indicate that AltDE is probably better suited to microaerophilic conditions and for the degradation of recalcitrant compounds such as urea. These, together with other features, and the distribution of this genotypic group, indicate that this microbe colonizes relatively large particles that sink rapidly to meso and bathypelagic depths. The genome of ATCC 27126 on the other hand has more potential for regulation (two component systems) and degrades more sugars and amino acids, which is consistent with a more transient particle attachment, as would be expected for lineages specialized in colonizing smaller particulate organic matter with much slower sinking rates. The genomic data are also consistent with a picture of incipient speciation driven by niche specialization.

Background: Colonisation of sessile eukaryotic host surfaces (e.g. invertebrates and seaweeds) by bacteria is common in the marine environment and is expected to create significant inter-species competition and other interactions. The bacterium Pseudoalteromonas tunicata is a successful competitor on marine surfaces owing primarily to its ability to produce a number of inhibitory molecules. As such P. tunicata has become a model organism for the studies into processes of surface colonisation and eukaryotic host-bacteria interactions., Methodology/principal Findings: To gain a broader understanding into the adaptation to a surface-associated life-style, we have sequenced and analysed the genome of P. tunicata and compared it to the genomes of closely related strains. We found that the P. tunicata genome contains several genes and gene clusters that are involved in the production of inhibitory compounds against surface competitors and secondary colonisers. Features of P. tunicata's oxidative stress response, iron scavenging and nutrient acquisition show that the organism is well adapted to high-density communities on surfaces. Variation of the P. tunicata genome is suggested by several landmarks of genetic rearrangements and mobile genetic elements (e.g. transposons, CRISPRs, phage). Surface attachment is likely to be mediated by curli, novel pili, a number of extracellular polymers and potentially other unexpected cell surface proteins. The P. tunicata genome also shows a utilisation pattern of extracellular polymers that would avoid a degradation of its recognised hosts, while potentially causing detrimental effects on other host types. In addition, the prevalence of recognised virulence genes suggests that P. tunicata has the potential for pathogenic interactions., Conclusions/significance: The genome analysis has revealed several physiological features that would provide P. tunciata with competitive advantage against other members of the surface-associated community. We have also identified properties that could mediate interactions with surfaces other than its currently recognised hosts. This together with the detection of known virulence genes leads to the hypothesis that P. tunicata maintains a carefully regulated balance between beneficial and detrimental interactions with a range of host surfaces.

Background: Polymerase chain reaction (PCR) is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment., Results: We have developed a fully integrated computational PCR primer design pipeline that plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions defined through a rich set of descriptors, such as Ensembl accessions and arbitrary genomic coordinates. Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving our sequencing success rate, which currently exceeds 95% for exons, we have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. We reveal the laboratory protocols and their associated, empirically determined computational parameters, as well as describe the novel computational methods which may benefit others in future primer design research., Conclusion: The high-throughput PCR primer design pipeline has been very successful in providing the basis for high-quality directed sequencing results and for minimizing costs associated with labor and reprocessing. The modular architecture of the primer design software has made it possible to readily integrate additional primer critique tests based on iterative feedback from the laboratory. As a result, the primer design software, coupled with the laboratory protocols, serves as a powerful tool for low and high-throughput primer design to enable successful directed sequencing.

Background: The picocyanobacterial genus Synechococcus occurs over wide oceanic expanses, having colonized most available niches in the photic zone. Large scale distribution patterns of the different Synechococcus clades (based on 16S rRNA gene markers) suggest the occurrence of two major lifestyles ('opportunists'/'specialists'), corresponding to two distinct broad habitats ('coastal'/'open ocean'). Yet, the genetic basis of niche partitioning is still poorly understood in this ecologically important group., Results: Here, we compare the genomes of 11 marine Synechococcus isolates, representing 10 distinct lineages. Phylogenies inferred from the core genome allowed us to refine the taxonomic relationships between clades by revealing a clear dichotomy within the main subcluster, reminiscent of the two aforementioned lifestyles. Genome size is strongly correlated with the cumulative lengths of hypervariable regions (or 'islands'). One of these, encompassing most genes encoding the light-harvesting phycobilisome rod complexes, is involved in adaptation to changes in light quality and has clearly been transferred between members of different Synechococcus lineages. Furthermore, we observed that two strains (RS9917 and WH5701) that have similar pigmentation and physiology have an unusually high number of genes in common, given their phylogenetic distance., Conclusion: We propose that while members of a given marine Synechococcus lineage may have the same broad geographical distribution, local niche occupancy is facilitated by lateral gene transfers, a process in which genomic islands play a key role as a repository for transferred genes. Our work also highlights the need for developing picocyanobacterial systematics based on genome-derived parameters combined with ecological and physiological data.

Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the world's oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the "leaves of the tree," between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Members of the gammaproteobacterial clade NOR5/OM60 regularly form an abundant part, up to 11%, of the bacterioplankton community in coastal systems during the summer months. Here, we report the nearly complete genome sequence of one cultured representative, Congregibacter litoralis strain KT71, isolated from North Sea surface water. Unexpectedly, a complete photosynthesis superoperon, including genes for accessory pigments, was discovered. It has a high sequence similarity to BAC clones from Monterey Bay [Beja O, Suzuki MT, Heidelberg JF, Nelson WC, Preston CM, et al. (2002) Nature 415:630-633], which also share a nearly identical gene arrangement. Although cultures of KT71 show no obvious pigmentation, bacteriochlorophyll a and spirilloxanthin-like carotenoids could be detected by HPLC analysis in cell extracts. The presence of two potential BLUF (blue light using flavin adenine dinucleotide sensors), one of which was found adjacent to the photosynthesis operon in the genome, indicates a light- and redox-dependent regulation of gene expression. Like other aerobic anoxygenic phototrophs (AAnPs), KT71 is able to grow neither anaerobically nor photoautotrophically. Cultivation experiments and genomic evidence show that KT71 needs organic substrates like carboxylic acids, oligopeptides, or fatty acids for growth. The strain grows optimally under microaerobic conditions and actively places itself in a zone of approximately 10% oxygen saturation. The genome analysis of C. litoralis strain KT71 identifies the gammaproteobacterial marine AAnPs, postulated based on BAC sequences, as members of the NOR5/OM60 clade. KT71 enables future experiments investigating the importance of this group of gammaproteobacterial AAnPs in coastal environments.