The microbial pan-genome

A decade after the beginning of the genomic era, the question of how genomics can describe a bacterial species has not been fully addressed. Experimental data have shown that in some species new genes are discovered even after sequencing the genomes of several strains. Mathematical modeling predicts that new genes will be discovered even after sequencing hundreds of genomes per species. Therefore, a bacterial species can be described by its pan-genome, which is composed of a "core genome" containing genes present in all strains, and a "dispensable genome" containing genes present in two or more strains and genes unique to single strains. Given that the number of unique genes is vast, the pan-genome of a bacterial species might be orders of magnitude larger than any single genome.     

Bacterial population genomics and infectious disease diagnostics

New sequencing technologies have made the production of bacterial genome sequences increasingly easy, and it can be confidently forecasted that vast genomic databases will be generated in the next few years. Here, we detail how collections of bacterial genomes from a particular species (population genomics libraries) have already been used to improve the design of several diagnostic assays for bacterial pathogens. Genome sequencing itself is also becoming more commonly used for epidemiological, forensic and clinical investigations. There is an opportunity for the further development of bioinformatic tools to bring even further value to bacterial diagnostic genomics.     

Microbial species delineation using whole genome sequences

Increased sequencing of microbial genomes has revealed that prevailing prokaryotic species assignments can be inconsistent with whole genome information for a significant number of species. The long-standing need for a systematic and scalable species assignment technique can be met by the genome-wide Average Nucleotide Identity (gANI) metric, which is widely acknowledged as a robust measure of genomic relatedness. In this work, we demonstrate that the combination of gANI and the alignment fraction (AF) between two genomes accurately reflects their genomic relatedness. We introduce an efficient implementation of AF,gANI and discuss its successful application to 86.5M genome pairs between 13,151 prokaryotic genomes assigned to 3032 species. Subsequently, by comparing the genome clusters obtained from complete linkage clustering of these pairs to existing taxonomy, we observed that nearly 18% of all prokaryotic species suffer from anomalies in species definition. Our results can be used to explore central questions such as whether microorganisms form a continuum of genetic diversity or distinct species represented by distinct genetic signatures. We propose that this precise and objective AF,gANI-based species definition: the MiSI (Microbial Species Identifier) method, be used to address previous inconsistencies in species classification and as the primary guide for new taxonomic species assignment, supplemented by the traditional polyphasic approach, as required.     

Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach

Advancements in next-generation sequencing technology have enabled whole genome re-sequencing in many species providing unprecedented discovery and characterization of molecular polymorphisms. There are limitations, however, to next-generation sequencing approaches for species with large complex genomes such as barley and wheat. Genotyping-by-sequencing (GBS) has been developed as a tool for association studies and genomics-assisted breeding in a range of species including those with complex genomes. GBS uses restriction enzymes for targeted complexity reduction followed by multiplex sequencing to produce high-quality polymorphism data at a relatively low per sample cost. Here we present a GBS approach for species that currently lack a reference genome sequence. We developed a novel two-enzyme GBS protocol and genotyped bi-parental barley and wheat populations to develop a genetically anchored reference map of identified SNPs and tags. We were able to map over 34,000 SNPs and 240,000 tags onto the Oregon Wolfe Barley reference map, and 20,000 SNPs and 367,000 tags on the Synthetic W9784 × Opata85 (SynOpDH) wheat reference map. To further evaluate GBS in wheat, we also constructed a de novo genetic map using only SNP markers from the GBS data. The GBS approach presented here provides a powerful method of developing high-density markers in species without a sequenced genome while providing valuable tools for anchoring and ordering physical maps and whole-genome shotgun sequence. Development of the sequenced reference genome(s) will in turn increase the utility of GBS data enabling physical mapping of genes and haplotype imputation of missing data. Finally, as a result of low per-sample costs, GBS will have broad application in genomics-assisted plant breeding programs.     

Poxvirus orthologous clusters: toward defining the minimum essential poxvirus genome

Increasingly complex bioinformatic analysis is necessitated by the plethora of sequence information currently available. A total of 21 poxvirus genomes have now been completely sequenced and annotated, and many more genomes will be available in the next few years. First, we describe the creation of a database of continuously corrected and updated genome sequences and an easy-to-use and extremely powerful suite of software tools for the analysis of genomes, genes, and proteins. These tools are available free to all researchers and, in most cases, alleviate the need for using multiple Internet sites for analysis. Further, we describe the use of these programs to identify conserved families of genes (poxvirus orthologous clusters) and have named the software suite POCs, which is available at www.poxvirus.org. Using POCs, we have identified a set of 49 absolutely conserved gene families-those which are conserved between the highly diverged families of insect-infecting entomopoxviruses and vertebrate-infecting chordopoxviruses. An additional set of 41 gene families conserved in chordopoxviruses was also identified. Thus, 90 genes are completely conserved in chordopoxviruses and comprise the minimum essential genome, and these will make excellent drug, antibody, vaccine, and detection targets. Finally, we describe the use of these tools to identify necessary annotation and sequencing updates in poxvirus genomes. For example, using POCs, we identified 19 genes that were widely conserved in poxviruses but missing from the vaccinia virus strain Tian Tan 1998 GenBank file. We have reannotated and resequenced fragments of this genome and verified that these genes are conserved in Tian Tan. The results for poxvirus genes and genomes are discussed in light of evolutionary processes.     

The Comprehensive Microbial Resource

One challenge presented by large-scale genome sequencing efforts is effective display of uniform information to the scientific community. The Comprehensive Microbial Resource (CMR) contains robust annotation of all complete microbial genomes and allows for a wide variety of data retrievals. The bacterial information has been placed on the Web at http://www.tigr.org/CMR for retrieval using standard web browsing technology. Retrievals can be based on protein properties such as molecular weight or hydrophobicity, GC-content, functional role assignments and taxonomy. The CMR also has special web-based tools to allow data mining using pre-run homology searches, whole genome dot-plots, batch downloading and traversal across genomes using a variety of datatypes.     

Mining the genomes of plant pathogenic bacteria: how not to drown in gigabases of sequence

Hundreds of bacterial genomes including the genomes of dozens of plant pathogenic bacteria have been sequenced. These genomes represent an invaluable resource for molecular plant pathologists. In this review, we describe different approaches that can be used for mining bacterial genome sequences and examples of how some of these approaches have been used to analyse plant pathogen genomes so far. We review how genomes can be mined one by one and how comparative genomics of closely related genomes releases the true power of genomics. Databases and tools useful for genome mining that are publicly accessible on the Internet are also described. Finally, the need for new databases and tools to efficiently mine today's plant pathogen genomes and hundreds more in the near future is discussed.     

Alternative splicing: a paradoxical qudo in eukaryotic genomes

One of the most remarkable observations stemming from the sequencing of genomes of diverse species is that the number of protein-coding genes in an organism does not correlate with its overall cellular complexity. Alternative splicing, a key mechanism for generating protein complexity, has been suggested as one of the major explanation for this discrepancy between the number of genes and genome complexity. Determining the extent and importance of alternative splicing required the confluence of critical advances in data acquisition, improved understanding of biological processes and the development of fast and accurate computational analysis tools. Although many model organisms have now been completely sequenced, we are still very far from understanding the exact frequency of alternative splicing from these sequenced genomes.This paper will highlight some recent progress and future challenges for functional genomics and bioinformatics in this rapidly developing area.     

Assembly and RNA‐free annotation of highly heterozygous genomes: The case of the thick‐billed murre (Uria lomvia)

Thanks to a dramatic reduction in sequencing costs followed by a rapid development of bioinformatics tools, genome assembly and annotation have become accessible to many researchers in recent years. Among tetrapods, birds have genomes that display many features that facilitate their assembly and annotation, such as small genome size, low number of repeats and highly conserved genomic structure. However, we found that high genomic heterozygosity could have a great impact on the quality of the genome assembly of the thick‐billed murre (Uria lomvia), an arctic colonial seabird. In this study, we tested the performance of three genome assemblers, ray /sscape , soapdenovo 2 and platanus , in assembling the highly heterozygous genome of the thick‐billed murre. Our results show that platanus , an assembler specifically designed for heterozygous genomes, outperforms the other two approaches and produces a highly contiguous (N50 = 15.8 Mb) and complete genome assembly (93% presence of genes from the Benchmarking Universal Single Copy Ortholog [BUSCO] gene set). Additionally, we annotated the thick‐billed murre genome using a homology‐based approach that takes advantage of the genomic resources available for birds and other taxa. Our study will be useful for those researchers who are approaching assembly and annotation of highly heterozygous genomes, or genomes of species of conservation concern, and/or who have limited financial resources.     

Current status and future possibilities of molecular genetics techniques in <Emphasis Type="Italic">Brassica napus</Emphasis>

As PCR methods have improved over the last 15 years, there has been an upsurge in the number of new DNA marker tools, which has allowed the generation of high-density molecular maps for all the key Brassica crop types. Biotechnology and molecular plant breeding have emerged as a significant tool for molecular understanding that led to a significant crop improvement in the Brassica napus species. Brassica napus possess a very complicated polyploidy-based genomics. The quantitative trait locus (QTL) is not sufficient to develop effective markers for trait introgression. In the coming years, the molecular marker techniques will be more effective to determine the whole genome impairing desired traits. Available genetic markers using the single-nucleotide sequence (SNP) technique and high-throughput sequencing are effective in determining the maps and genome polymorphisms amongst candidate genes and allele interactions. High-throughput sequencing and gene mapping techniques are involved in discovering new alleles and gene pairs, serving as a bridge between the gene map and genome evaluation. The decreasing cost for DNA sequencing will help in discovering full genome sequences with less resources and time. This review describes (1) the current use of integrated approaches, such as molecular marker technologies, to determine genome arrangements and interspecific outcomes combined with cost-effective genomes to increase the efficiency in prognostic breeding efforts. (2) It also focused on functional genomics, proteomics and field-based breeding practices to achieve insight into the genetics underlying both simple and complex traits in canola.     

A White Paper on Nematode Comparative Genomics

In response to the new opportunities for genome sequencing and comparative genomics, the Society of Nematology (SON) formed a committee to develop a white paper in support of the broad scientific needs associated with this phylum and interests of SON members. Although genome sequencing is expensive, the data generated are unique in biological systems in that genomes have the potential to be complete (every base of the genome can be accounted for), accurate (the data are digital and not subject to stochastic variation), and permanent (once obtained, the genome of a species does not need to be experimentally re-sampled). The availability of complete, accurate, and permanent genome sequences from diverse nematode species will underpin future studies into the biology and evolution of this phylum and the ecological associations (particularly parasitic) nematodes have with other organisms. We anticipate that upwards of 100 nematode genomes will be solved to varying levels of completion in the coming decade and suggest biological and practical considerations to guide the selection of the most informative taxa for sequencing.     

Comparison of genomic DNA sequences: solved and unsolved problems

MOTIVATION: The DNA sequences of entire genomes are being determined at a rapid rate. Whereas initial genome sequencing efforts were for organisms chosen to be widely spaced in the tree of life, there is a growing emphasis on projects to sequence a species that is sufficiently similar to an already-sequenced species to allow direct comparison of those two DNA sequences. This and other changes in genome sequencing strategies have created a strong need for new methods to compare genomic sequences. RESULTS: We sketch the current state of software for comparing genomic DNA sequences and outline research directions that we believe are likely to result in important advances in practice.     

Plant genomics in Africa: present and prospects

Plants are the world’s most consumed goods. They are of high economic value and bring many health benefits. In most countries in Africa, the supply and quality of food will rise to meet the growing population’s increasing demand. Genomics and other biotechnology tools offer the opportunity to improve subsistence crops and medicinal herbs in the continent. Significant advances have been made in plant genomics, which have enhanced our knowledge of the molecular processes underlying both plant quality and yield. The sequencing of complex genomes of African plant species, facilitated by the continuously evolving next-generation sequencing technologies and advanced bioinformatics approaches, has provided new opportunities for crop improvement. This review summarizes the achievements of genome sequencing projects of endemic African plants in the last two decades. We also present perspectives and challenges for future plant genomic studies that will accelerate important plant breeding programs for African communities. These challenges include a lack of basic facilities, a lack of sequencing and bioinformatics facilities, and a lack of skills to design genomics studies. However, it is imperative to state that African countries have become key players in the plant genome revolution and genome derived-biotechnology. Therefore, African governments should invest in public plant genomics research and applications, establish bioinformatics platforms and training programs, and stimulate university and industry partnerships to fully deploy plant genomics, particularly in the fields of agriculture and medicine.     

Genetic and phenotypic characterization of Phaeodactylum tricornutum (Bacillariophyceae) accessions1

In the last few years, genome‐based studies in diatoms have received a major boost following the genome sequencing of the centric species Thalassiosira pseudonana Hasle et Heimdal and the pleiomorphic raphid pennate diatom Phaeodactylum tricornutum Bohlin. In addition, molecular tools, such as genetic transformation, have been developed for both species. Despite these molecular advances, relatively little is known regarding the genetic diversity of the available strains of these diatoms. In this study, we have compiled a historical summary of the known P. tricornutum species resources and have provided a genetic and phenotypic overview of 10 different axenic strains. Examination of intraspecies genetic diversity based on internal transcribed spacer 2 (ITS2) sequence and amplified fragment length polymorphism (AFLP) analyses indicate four different genotypes. Seven strains are predominantly fusiform, whereas one strain is predominantly oval, and another is predominantly triradiate. Another is defined as a tropical strain because it appears better acclimated to growth at higher temperatures. Observations in the natural environment indicate that P. tricornutum is a coastal marine diatom that is able to adapt to unstable environments, such as estuaries and rock pools. Because it has rarely been noted in nature, we have developed specific primers to amplify ITS2 sequences and have successfully identified it in environmental samples. These resources should become useful tools for the diatom community when combined with the whole genome sequence and will open up a range of new possibilities for experimental investigations that can exploit the genotypic and phenotypic characteristics described.     

What's buzzing? Mosquito genomics and transgenic mosquitoes

Genome projects and associated technologies are now being established for mosquito species that are vectors of human disease. The recent announcement of an award by the National Institute of Allergy and Infectious Diseases (NIAID) to Celera Genomics to sequence the Anopheles gambiae genome will further accelerate the completion of the sequencing of this genome. Completion of the An. gambiae sequence will mean that the genomes of all three organisms involved in the transmission of falciparum malaria--the mosquito, the parasite, and the human--will have been sequenced. This will greatly facilitate the identification of genes and pathways involved in the transmission of malaria. The recent genetic transformation of An. gambiae with the piggyBac transposable element and the transformation of another important malarial vector, Anopheles stephensi using the Minos element, now provide researchers with powerful tools with which to genetically manipulate these medically important vector species. Here we review the recent progress made in the extension of contemporary tools of modern genetics and genomics into these medically important insects.     

The maize genome as a model for efficient sequence analysis of large plant genomes

The genomes of flowering plants vary in size from about 0.1 to over 100 gigabase pairs (Gbp), mostly because of polyploidy and variation in the abundance of repetitive elements in intergenic regions. High-quality sequences of the relatively small genomes of Arabidopsis (0.14 Gbp) and rice (0.4 Gbp) have now been largely completed. The sequencing of plant genomes that have a more representative size (the mean for flowering plant genomes is 5.6 Gbp) has been seen as a daunting task, partly because of their size and partly because of the numerous highly conserved repeats. Nevertheless, creative strategies and powerful new tools have been generated recently in the plant genetics community, so that sequencing large plant genomes is now a realistic possibility. Maize (2.4-2.7 Gbp) will be the first gigabase-size plant genome to be sequenced using these novel approaches. Pilot studies on maize indicate that the new gene-enrichment, gene-finishing and gene-orientation technologies are efficient, robust and comprehensive. These strategies will succeed in sequencing the gene-space of large genome plants, and in locating all of these genes and adjacent sequences on the genetic and physical maps.     

Legume genomes: more than peas in a pod

A growing array of sequence-based tools is helping to reveal the organization, evolution and syntenic relationships of legume genomes. The results indicate that legumes form a coherent taxonomic group with frequent and widespread macro- and microsynteny. This is good news for two model legume systems, Medicago truncatula and Lotus japonicus. Indeed, both models have recently been used to clone and characterize genes for nodulation-related receptors that were originally described in legumes with more complex genomes. Studies of legume genomes have also provided insight into genome size, gene clustering, genome duplications and repetitive elements. To understand legume genomes better, it will be necessary to develop tools for studying under-represented taxa beyond the relatively small group of economically important species that have been examined so far.