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PubMed2005

Genomics and public health: development of Web-based training tools for increasing genomic awareness.

In 2001, the Centers for Disease Control and Prevention funded three Centers for Genomics and Public Health to develop training tools for increasing genomic awareness. Over the past three years, the centers, working together with the Centers for Disease Control and Prevention's Office of Genomics and Disease Prevention, have developed tools to increase awareness of the impact genomics will have on public health practice, to provide a foundation for understanding basic genomic advances, and to translate the relevance of that information to public health practitioners' own work. These training tools serve to communicate genomic advances and their potential for integration into public heath practice. This paper highlights two of these training tools: 1) Genomics for Public Health Practitioners: The Practical Application of Genomics in Public Health Practice, a Web-based introduction to genomics, and 2) Six Weeks to Genomic Awareness, an in-depth training module on public health genomics. This paper focuses on the processes and collaborative efforts by which these live presentations were developed and delivered as Web-based training sessions.

Costs and Cost Analysis↗

PubMed2005

Genome trees and the nature of genome evolution.

Genome trees are a means to capture the overwhelming amount of phylogenetic information that is present in genomes. Different formalisms have been introduced to reconstruct genome trees on the basis of various aspects of the genome. On the basis of these aspects, we separate genome trees into five classes: (a) alignment-free trees based on statistic properties of the genome, (b) gene content trees based on the presence and absence of genes, (c) trees based on chromosomal gene order, (d) trees based on average sequence similarity, and (e) phylogenomics-based genome trees. Despite their recent development, genome tree methods have already had some impact on the phylogenetic classification of bacterial species. However, their main impact so far has been on our understanding of the nature of genome evolution and the role of horizontal gene transfer therein. An ideal genome tree method should be capable of using all gene families, including those containing paralogs, in a phylogenomics framework capitalizing on existing methods in conventional phylogenetic reconstruction. We expect such sophisticated methods to help us resolve the branching order between the main bacterial phyla.

Evolution, Molecular↗

PubMed2003

Identification of genomic islands in the genome of Bacillus cereus by comparative analysis with Bacillus anthracis.

Horizontal gene transfer has been recognized as a universal event throughout bacterial evolution. The availability of both complete genome sequences of Bacillus cereus and B. anthracis provides the possibility to perform comparative analysis based on their genomes. By using a windowless method to display the distribution of the genomic GC content of B. cereus and B. anthracis, we have found three genomic islands in the genome of B. cereus, i.e., BCGI-1, BCGI-2, and BCGI-3, respectively, which are absent in the genome of B. anthracis. All the genomic islands have abrupt changes in GC content compared with that of surrounding regions. BCGI-1 has many conserved features of genomic islands, e.g., a Val-tRNA gene is utilized as the integration site, and a site-specific recombinase gene is located at the 3' end. BCGI-2 has a large percentage of phage protein, suggesting a phage-related recombination is involved. BCGI-3 contains a ferric anguibactin transport system, which is likely to be involved in the iron transport that enables the bacterium to overcome the iron limitation in the host. In addition, BCGI-3 also contains a cluster of genes related to lantibiotics, which may play a role during the evolution of the genome. Furthermore, the integrations of the genomic islands, BCGI-1 and BCGI-3, result in deletions of DNA sequence fragments; therefore, such integrations lead to both gene gain and gene loss simultaneously.

Bacillus anthracis↗

PubMed2006

Using partial genomic fosmid libraries for sequencing complete organellar genomes.

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However; for some organisms, it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.

DNA↗

PubMed2004

"Harnessing genomics to improve health in India" - an executive course to support genomics policy.

BACKGROUND: The benefits of scientific medicine have eluded millions in developing countries and the genomics revolution threatens to increase health inequities between North and South. India, as a developing yet also industrialized country, is uniquely positioned to pioneer science policy innovations to narrow the genomics divide. Recognizing this, the Indian Council of Medical Research and the University of Toronto Joint Centre for Bioethics conducted a Genomics Policy Executive Course in January 2003 in Kerala, India. The course provided a forum for stakeholders to discuss the relevance of genomics for health in India. This article presents the course findings and recommendations formulated by the participants for genomics policy in India. METHODS: The course goals were to familiarize participants with the implications of genomics for health in India; analyze and debate policy and ethical issues; and develop a multi-sectoral opinion leaders' network to share perspectives. To achieve these goals, the course brought together representatives of academic research centres, biotechnology companies, regulatory bodies, media, voluntary, and legal organizations to engage in discussion. Topics included scientific advances in genomics, followed by innovations in business models, public sector perspectives, ethics, legal issues and national innovation systems. RESULTS: Seven main recommendations emerged: increase funding for healthcare research with appropriate emphasis on genomics; leverage India's assets such as traditional knowledge and genomic diversity in consultation with knowledge-holders; prioritize strategic entry points for India; improve industry-academic interface with appropriate incentives to improve public health and the nation's wealth; develop independent, accountable, transparent regulatory systems to ensure that ethical, legal and social issues are addressed for a single entry, smart and effective system; engage the public and ensure broad-based input into policy setting; ensure equitable access of poor to genomics products and services; deliver knowledge, products and services for public health. A key outcome of the course was the internet-based opinion leaders' network - the Indian Genome Policy Forum - a multi-stakeholder forum to foster further discussion on policy. CONCLUSION: We expect that the process that has led to this network will serve as a model to establish similar Science and Technology policy networks on regional levels and eventually on a global level.

Journal Article↗

PubMed2001

Genomic in situ hybridization (GISH) discriminates between the A and the B genomes in diploid and tetraploid Setaria species.

Genomic in situ hybridization (GISH) was used to investigate genomic relationships between different Setaria species of the foxtail millet gene pool (S. italica) and one interspecific F1 hybrid. The GISH patterns obtained on the two diploid species S. viridis (genome A) and S. adhaerans (genome B), and on their F1 hybrid showed clear differentiation between these two genomes except at the nucleolar organizing regions. Similar GISH patterns allowed differentiation of S. italica from S. adhaerans. However, GISH patterns did not distinguish between the genomes of S. italica and its putative wild ancestor S. viridis. GISH was also applied to polyploid Setaria species and enabled confirmation of the assumed allotetraploid nature of S. faberii and demonstration that both S. verticillata and S. verticillata var. ambigua were also allotetraploids. All these tetraploid species contained two sets of 18 chromosomes each, one from genome A and the other from genome B. Only one polyploid species, S. pumila, was shown to bear an unknown genomic composition that is not closely related either to genome A or to genome B.

Animals↗

PubMed2005

How can we use genomics to improve cereals with rice as a reference genome?

Rice serves as a model crop for cereal genomics. The availability of complete genome sequences, together with various genomic resources available for both rice and Arabidopsis, have revolutionized our understanding of the genetic make-up of crop plants. Both macrocolinearity revealed by comparative mapping and microcolinearity revealed by sequence comparisons among the grasses indicate that sequencing and functional analysis of the rice genome will have a significant impact on other cereals in terms of both genomic studies and crop improvement. The availability of mutants, introgression libraries, and advanced transformation techniques make functional genomics in rice and other cereals more manageable than ever before. A wide array of genetic markers, including anchor markers for comparative mapping, SSRs and SNPs are widely used in genetic mapping, germplasm evaluation and marker assisted selection. An integrated database that combines genome information for rice and other cereals is key to the effective utilization of all genomics resources for cereal improvement. To maximize the potential of genomics for plant breeding, experiments must be further miniaturized and costs must be reduced. Many techniques, including targeted gene disruption or allele substitution, insertional mutagenesis, RNA interference and homologous recombination, need to be refined before they can be widely used in functional genomic analysis and plant breeding.

Chromosome Mapping↗

PubMed2025

Combinatorial genome engineering of pseudorabies virus Bartha by developing a reverse genetic system based on three overlapping genomic segments.

INTRODUCTION: The 138-kilobase genome of pseudorabies virus vaccine strain Bartha K61 harbors many nonessential genes for replication and exhibits remarkable capacity for incorporating foreign genes for therapeutic applications. However, the large size of the Bartha genome complicates its efficient engineering. OBJECTIVES: Development of a reverse genetic system for pseudorabies virus Bartha based on three overlapping genomic segments to facilitate multiplex genome engineering. METHODS: The 138-kb genome of Bartha was split into three overlapping segments (42 kb, 43 kb, and 53 kb), each cloned in a bacterial artificial chromosome (BAC) to facilitate genome engineering. The infectious virus was reconstituted by transfecting the 3 genomic fragments released from the BACs into Vero cells in which a complete virus genome was assembled using 2-kb overlaps between adjacent pieces. RESULTS: Employing the reverse genetic system, we individually deleted 15 candidate nonessential genes and confirmed that 10 were dispensable for viral growth in cell culture. Deletion of 7 nonessential genes had no impact on viral growth, whereas UL47 deletion reduced viral growth rate and deletions of UL44, UL47, or US3 resulted in smaller viral plaques. A total of 45 viral genomes with double deletions of nonessential genes were constructed, among which 22 were successfully rescued into infectious virions. Fifteen double-deletion mutant viruses had a viral titer comparable with the wild-type Bartha, while the remaining 7 showed a lower titer. Additionally, expressions of the mNeonGreen reporter gene at nonessential gene loci were evaluated. Cells infected with recombinant viruses carrying mNeonGreen at 8 loci showed strong green fluorescence, whereas those with mNeonGreen at 2 loci exhibited very weak fluorescence. CONCLUSION: The reverse genetic system developed in this study enables rapid and combinatorial engineering of viruses with the large DNA genome, and will accelerate development of large DNA virus-based therapeutics including live-attenuated vaccines, vector vaccines, and oncolytic herpesviruses.

Herpesvirus 1, Suid↗

PubMed2002

Rice genome organization: the centromere and genome interactions.

Over the last decade, many varied resources have become available for genome studies in rice. These resources include over 4000 DNA markers, several bacterial artificial chromosome (BAC) libraries, P-1 derived artificial chromosome (PAC) libraries and yeast artificial chromosome (YAC) libraries (genomic DNA clones, filters and end-sequences), retrotransposon tagged lines, and many chemical and irradiated mutant lines. Based on these, high-density genetic maps, cereal comparative maps, YAC and BAC physical maps, and quantitative trait loci (QTL) maps have been constructed, and 93 % of the genome has also been sequenced. These data have revealed key features of the genetic and physical structure of the rice genome and of the evolution of cereal chromosomes. This Botanical Briefing examines aspects of how the rice genome is organized structurally, functionally and evolutionarily. Emphasis is placed on the rice centromere, which is composed of long arrays of centromere-specific repetitive sequences. Differences and similarities amongst various cereal centromeres are detailed. These indicate essential features of centromere function. Another view of various kinds of interactive relationships within and between genomes, which could play crucial roles in genome organization and evolution, is also introduced. Constructed genetic and physical maps indicate duplication of chromosomal segments and spatial association between specific chromosome regions. A genome-wide survey of interactive genetic loci has identified various reproductive barriers that may drive speciation of the rice genome. The significance of these findings in genome organization and evolution is discussed.

Centromere↗

PubMed2025

MutBERT: probabilistic genome representation improves genomics foundation models.

MOTIVATION: Understanding the genomic foundation of human diversity and disease requires models that effectively capture sequence variation, such as single nucleotide polymorphisms (SNPs). While recent genomic foundation models have scaled to larger datasets and multi-species inputs, they often fail to account for the sparsity and redundancy inherent in human population data, such as those in the 1000 Genomes Project. SNPs are rare in humans, and current masked language models (MLMs) trained directly on whole-genome sequences may struggle to efficiently learn these variations. Additionally, training on the entire dataset without prioritizing regions of genetic variation results in inefficiencies and negligible gains in performance. RESULTS: We present MutBERT, a probabilistic genome-based masked language model that efficiently utilizes SNP information from population-scale genomic data. By representing the entire genome as a probabilistic distribution over observed allele frequencies, MutBERT focuses on informative genomic variations while maintaining computational efficiency. We evaluated MutBERT against DNABERT-2, various versions of Nucleotide Transformer, and modified versions of MutBERT across multiple downstream prediction tasks. MutBERT consistently ranked as one of the top-performing models, demonstrating that this novel representation strategy enables better utilization of biobank-scale genomic data in building pretrained genomic foundation models. AVAILABILITY AND IMPLEMENTATION: https://github.com/ai4nucleome/mutBERT.

Humans↗

PubMed2003

The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics.

The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.

Animals↗

PubMed2006

Sequencing a new target genome: the Boophilus microplus (Acari: Ixodidae) genome project.

The southern cattle tick, Boophilus microplus (Canestrini), causes annual economic losses in the hundreds of millions of dollars to cattle producers throughout the world, and ranks as the most economically important tick from a global perspective. Control failures attributable to the development of pesticide resistance have become commonplace, and novel control technologies are needed. The availability of the genome sequence will facilitate the development of these new technologies, and we are proposing sequencing to a 4-6X draft coverage. Many existing biological resources are available to facilitate a genome sequencing project, including several inbred laboratory tick strains, a database of approximately 45,000 expressed sequence tags compiled into a B. microplus Gene Index, a bacterial artificial chromosome (BAC) library, an established B. microplus cell line, and genomic DNA suitable for library synthesis. Collaborative projects are underway to map BACs and cDNAs to specific chromosomes and to sequence selected BAC clones. When completed, the genome sequences from the cow, B. microplus, and the B. microplus-borne pathogens Babesia bovis and Anaplasma marginale will enhance studies of host-vector-pathogen systems. Genes involved in the regeneration of amputated tick limbs and transitions through developmental stages are largely unknown. Studies of these and other interesting biological questions will be advanced by tick genome sequence data. Comparative genomics offers the prospect of new insight into many, perhaps all, aspects of the biology of ticks and the pathogens they transmit to farm animals and people. The B. microplus genome sequence will fill a major gap in comparative genomics: a sequence from the Metastriata lineage of ticks. The purpose of the article is to synergize interest in and provide rationales for sequencing the genome of B. microplus and for publicizing currently available genomic resources for this tick.

Animals↗

PubMed1995

Size-selected genomic libraries: the distribution and size-fractionation of restricted genomic DNA fragments by gel electrophoresis.

By using one-dimensional genome scanning, it is possible to directly identify the restricted genomic DNA fragment that reflects the site of genetic change. The subsequent strategies to obtain the molecular clones of the corresponding restriction fragment are usually as follows: (i) the restriction of a mass quantity of an appropriate genomic DNA, (ii) the size-fractionation of the restricted DNA on a preparative electrophoresis gel in order to enrich the corresponding restriction fragment, (iii) the construction of the size-selected libraries from the fractionated genomic DNA, and (iv) the screening of the library to obtain an objective clone which is identified on the analytical genome scanning gel. A knowledge of the size distribution pattern of restriction fragments of the genomic DNA makes it possible to calculate the heterogeneity or complexity of the restriction fragment in each size-fraction. This manuscript first describes the distribution of the restriction fragments with respect to their length. Some examples of the practical application of this theory to genome scanning is then discussed using presumptive genome scanning gels. The way to calculate such DNA complexities in the prepared size-fractionated samples is also demonstrated. Such information should greatly facilitate the design of experimental strategies for the cloning of a certain size of genomic DNA after digestion with restriction enzyme(s) as is the case with genome scanning.

Animals↗

PubMed2005

Testing of a whole genome PCR scanning approach to identify genomic variability in four different species of lactic acid bacteria.

Genomes can be markedly heterogeneous in conspecific bacterial strains. Genome sequences can be used to analyze genome plasticity via a PCR(2) (plasticity of chromosome revealed by PCR) approach. Small-sized chromosomes can indeed be fully amplified by long-range PCR with a set of primers designed using a reference strain and then applied to several other strains. Analysis of the resulting patterns can reveal genome plasticity. GenoFrag, a software package for the design of primers optimized for PCR(2) [N. Ben Zakour, M. Gautier, R. Andonov, D. Lavenier, M.F. Cochet, P. Veber, A. Sorokin, Y. Le Loir, GenoFrag: Software to design primers optimized for whole genome scanning by long-range PCR amplification, Nucleic Acids Res. 32 (2004) 17-24] was developed for the analysis of bacterial genome plasticity by whole genome amplification in approximately 10-kb-long fragments. By applying GenoFrag, we provide herewith evidence that genome plasticity can be analyzed in lactic acid bacteria using a PCR(2) approach. The genome sequences of Lactococcus lactis IL1403, Lactobacillus plantarum WCFS1, Lactobacillus bulgaricus ATCC11842 and Bifidobacterium longum NCC2705 were used to design four sets of primers. Each set was evaluated in silico to check that it ensured optimum coverage of the bacterial chromosome. To validate the primers generated by GenoFrag, a subset of primers was successfully used in LR-PCR experiments on genomic DNA from four L. bulgaricus strains.

Bacterial Typing Techniques↗

PubMed1993

Characterization of the pufferfish (Fugu) genome as a compact model vertebrate genome.

Cloning and sequencing techniques now allow us to characterize genes directly instead of having to deduce their properties from their effects. This new genetics reaches its apotheosis in the plan to obtain the complete DNA sequence of the human genome, but this is far beyond the capacity of present sequencing methods. Small 'model' genomes, 'such as those of Escherichia coli (4.7 megabases (Mb) and yeast (14 Mb), or even those of Caenorhabditis elegans (100 Mb) and Drosophila (165 Mb), are better scaled to existing technology. The yeast genome will contain genes with functions common to all eukaryotic cells, and those of simple multicellular organisms may throw light on the genetic specification of more complex functions. However, vertebrates differ in their morphology and development, so the ideal model would be a vertebrate genome of minimum size and complexity but with maximum homology to the human genome. Here we report the characterization of the small genome (400 Mb) of the tetraodontoid fish, Fugu rubripes. A random sequencing approach supported by gene probing shows that the haploid genome contains 400 Mb of DNA, of which more that 90% is unique. This genome is 7.5 times smaller than the human genome and because it has a similar gene repertoire it is the best model genome for the discovery of human genes.

Animals↗

PubMed1996

Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA.

Genetic analysis of limiting quantities of genomic DNA play an important role in DNA forensics, paleoarcheology, genetic disease diagnosis, genetic linkage analysis, and genetic diversity studies. We have tested the ability of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to amplify picogram quantities of human genomic DNA for the purpose of increasing the amount of template for genotyping with microsatellite repeat markers. DNA was uniformly amplified at a large number of typable loci throughout the human genome with starting template DNAs from as little as 15 pg to as much as 400 ng. A much greater-fold enrichment was seen for the smaller genomic DOP-PCRs. All markers tested were amplified from starting genomic DNAs in the range of 0.6-40 ng with amplifications of 200- to 600-fold. The DOP-PCR-amplified genomic DNA was an excellent and reliable template for genotyping with microsatellites, which give distinct bands with no increase in stutter artifact on di-, tri-, and tetranucleotide repeats. There appears to be equal amplification of genomic DNA from 55 of 55 tested discrete microsatellites implying near complete coverage of the human genome. Thus, DOP-PCR appears to allow unbiased, hundreds-fold whole genome amplification of human genomic DNA for genotypic analysis.

DNA↗

PubMed2004

Comparative architectures of mammalian and chicken genomes reveal highly variable rates of genomic rearrangements across different lineages.

Molecular evolution studies are usually based on the analysis of individual genes and thus reflect only small-range variations in genomic sequences. A complementary approach is to study the evolutionary history of rearrangements in entire genomes based on the analysis of gene orders. The progress in whole genome sequencing provides an unprecedented level of detailed sequence data to infer genome rearrangements through comparative approaches. The comparative analysis of recently sequenced rodent genomes with the human genome revealed evidence for a larger number of rearrangements than previously thought and led to the reconstruction of the putative genomic architecture of the murid rodent ancestor, while the architecture of the ancestral mammalian genome and the rate of rearrangements in the human lineage remained unknown. Sequencing the chicken genome provides an opportunity to reconstruct the architecture of the ancestral mammalian genome by using chicken as an outgroup. Our analysis reveals a very low rate of rearrangements and, in particular, interchromosomal rearrangements in chicken, in the early mammalian ancestor, or in both. The suggested number of interchromosomal rearrangements between the mammalian ancestor and chicken, during an estimated 500 million years of evolution, only slightly exceeds the number of interchromosomal rearrangements that happened in the mouse lineage, over the course of about 87 million years.

Animals↗

PubMed2001

Conservation of microstructure between a sequenced region of the genome of rice and multiple segments of the genome of Arabidopsis thaliana.

The nucleotide sequence was determined for a 340-kb segment of rice chromosome 2, revealing 56 putative protein-coding genes. This represents a density of one gene per 6.1 kb, which is higher than was reported for a previously sequenced segment of the rice genome. Sixteen of the putative genes were supported by matches to ESTs. The predicted products of 29 of the putative genes showed similarity to known proteins, and a further 17 genes showed similarity only to predicted or hypothetical proteins identified in genome sequence data. The region contains a few transposable elements: one retrotransposon, and one transposon. The segment of the rice genome studied had previously been identified as representing a part of rice chromosome 2 that may be homologous to a segment of Arabidopsis chromosome 4. We confirmed the conservation of gene content and order between the two genome segments. In addition, we identified a further four segments of the Arabidopsis genome that contain conserved gene content and order. In total, 22 of the 56 genes identified in the rice genome segment were represented in this set of Arabidopsis genome segments, with at least five genes present, in conserved order, in each segment. These data are consistent with the hypothesis that the Arabidopsis genome has undergone multiple duplication events. Our results demonstrate that conservation of the genome microstructure can be identified even between monocot and dicot species. However, the frequent occurrence of duplication, and subsequent microstructure divergence, within plant genomes may necessitate the integration of subsets of genes present in multiple redundant segments to deduce evolutionary relationships and identify orthologous genes.

Arabidopsis↗