High-coverage Genome Research Articles

Comrad: detection of expressed rearrangements by integrated analysis of RNA-Seq and low coverage genome sequence data

Comrad is a novel algorithmic framework for the integrated analysis of RNA-Seq and whole genome shotgun sequencing (WGSS) data for the purposes of discovering genomic rearrangements and aberrant transcripts. The Comrad framework leverages the advantages of both RNA-Seq and WGSS data, providing accurate classification of rearrangements as expressed or not expressed and accurate classification of the genomic or non-genomic origin of aberrant transcripts. A major benefit of Comrad is its ability to accurately identify aberrant transcripts and associated rearrangements using low coverage genome data. As a result, a Comrad analysis can be performed at a cost comparable to that of two RNA-Seq experiments, significantly lower than an analysis requiring high coverage genome data. We have applied Comrad to the discovery of gene fusions and read-throughs in prostate cancer cell line C4-2, a derivative of the LNCaP cell line with androgen-independent characteristics. As a proof of concept, we have rediscovered in the C4-2 data 4 of the 6 fusions previously identified in LNCaP. We also identified six novel fusion transcripts and associated genomic breakpoints, and verified their existence in LNCaP, suggesting that Comrad may be more sensitive than previous methods that have been applied to fusion discovery in LNCaP. We show that many of the gene fusions discovered using Comrad would be difficult to identify using currently available techniques. A C++ and Perl implementation of the method demonstrated in this article is available at http://compbio.cs.sfu.ca/.

Nonsynonymous substitution rate (Ka) is a relatively consistent parameter for defining fast-evolving and slow-evolving protein-coding genes

BackgroundMammalian genome sequence data are being acquired in large quantities and at enormous speeds. We now have a tremendous opportunity to better understand which genes are the most variable or conserved, and what their particular functions and evolutionary dynamics are, through comparative genomics.ResultsWe chose human and eleven other high-coverage mammalian genome data–as well as an avian genome as an outgroup–to analyze orthologous protein-coding genes using nonsynonymous (Ka) and synonymous (Ks) substitution rates. After evaluating eight commonly-used methods of Ka and Ks calculation, we observed that these methods yielded a nearly uniform result when estimating Ka, but not Ks (or Ka/Ks). When sorting genes based on Ka, we noticed that fast-evolving and slow-evolving genes often belonged to different functional classes, with respect to species-specificity and lineage-specificity. In particular, we identified two functional classes of genes in the acquired immune system. Fast-evolving genes coded for signal-transducing proteins, such as receptors, ligands, cytokines, and CDs (cluster of differentiation, mostly surface proteins), whereas the slow-evolving genes were for function-modulating proteins, such as kinases and adaptor proteins. In addition, among slow-evolving genes that had functions related to the central nervous system, neurodegenerative disease-related pathways were enriched significantly in most mammalian species. We also confirmed that gene expression was negatively correlated with evolution rate, i.e. slow-evolving genes were expressed at higher levels than fast-evolving genes. Our results indicated that the functional specializations of the three major mammalian clades were: sensory perception and oncogenesis in primates, reproduction and hormone regulation in large mammals, and immunity and angiotensin in rodents.ConclusionOur study suggests that Ka calculation, which is less biased compared to Ks and Ka/Ks, can be used as a parameter to sort genes by evolution rate and can also provide a way to categorize common protein functions and define their interaction networks, either pair-wise or in defined lineages or subgroups. Evaluating gene evolution based on Ka and Ks calculations can be done with large datasets, such as mammalian genomes.ReviewersThis article has been reviewed by Drs. Anamaria Necsulea (nominated by Nicolas Galtier), Subhajyoti De (nominated by Sarah Teichmann) and Claus O. Wilke.

MicroRNAome of Porcine Pre- and Postnatal Development

The domestic pig is of enormous agricultural significance and valuable models for many human diseases. Information concerning the pig microRNAome (miRNAome) has been long overdue and elucidation of this information will permit an atlas of microRNA (miRNA) regulation functions and networks to be constructed. Here we performed a comprehensive search for porcine miRNAs on ten small RNA sequencing libraries prepared from a mixture of tissues obtained during the entire pig lifetime, from the fetal period through adulthood. The sequencing results were analyzed using mammalian miRNAs, the precursor hairpins (pre-miRNAs) and the first release of the high-coverage porcine genome assembly (Sscrofa9, April 2009) and the available expressed sequence tag (EST) sequences. Our results extend the repertoire of pig miRNAome to 867 pre-miRNAs (623 with genomic coordinates) encoding for 1,004 miRNAs, of which 777 are unique. We preformed real-time quantitative PCR (q-PCR) experiments for selected 30 miRNAs in 47 tissue-specific samples and found agreement between the sequencing and q-PCR data. This broad survey provides detailed information about multiple variants of mature sequences, precursors, chromosomal organization, development-specific expression, and conservation patterns. Our data mining produced a broad view of the pig miRNAome, consisting of miRNAs and isomiRs and a wealth of information of pig miRNA characteristics. These results are prelude to the advancement in pig biology as well the use of pigs as model organism for human biological and biomedical studies.

A high-density Diversity Arrays Technology (DArT) microarray for genome-wide genotyping in Eucalyptus

BackgroundA number of molecular marker technologies have allowed important advances in the understanding of the genetics and evolution of Eucalyptus, a genus that includes over 700 species, some of which are used worldwide in plantation forestry. Nevertheless, the average marker density achieved with current technologies remains at the level of a few hundred markers per population. Furthermore, the transferability of markers produced with most existing technology across species and pedigrees is usually very limited. High throughput, combined with wide genome coverage and high transferability are necessary to increase the resolution, speed and utility of molecular marker technology in eucalypts. We report the development of a high-density DArT genome profiling resource and demonstrate its potential for genome-wide diversity analysis and linkage mapping in several species of Eucalyptus.FindingsAfter testing several genome complexity reduction methods we identified the PstI/TaqI method as the most effective for Eucalyptus and developed 18 genomic libraries from PstI/TaqI representations of 64 different Eucalyptus species. A total of 23,808 cloned DNA fragments were screened and 13,300 (56%) were found to be polymorphic among 284 individuals. After a redundancy analysis, 6,528 markers were selected for the operational array and these were supplemented with 1,152 additional clones taken from a library made from the E. grandis tree whose genome has been sequenced. Performance validation for diversity studies revealed 4,752 polymorphic markers among 174 individuals. Additionally, 5,013 markers showed segregation when screened using six inter-specific mapping pedigrees, with an average of 2,211 polymorphic markers per pedigree and a minimum of 859 polymorphic markers that were shared between any two pedigrees.ConclusionsThis operational DArT array will deliver 1,000-2,000 polymorphic markers for linkage mapping in most eucalypt pedigrees and thus provide high genome coverage. This array will also provide a high-throughput platform for population genetics and phylogenetics in Eucalyptus. The transferability of DArT across species and pedigrees is particularly valuable for a large genus such as Eucalyptus and will facilitate the transfer of information between different studies. Furthermore, the DArT marker array will provide a high-resolution link between phenotypes in populations and the Eucalyptus reference genome, which will soon be completed.

Abstract 93: COSMIC: The catalogue of somatic mutations in cancer receives full genome variant annotations

Abstract COSMIC, the Catalogue Of Somatic Mutations In Cancer (http://www.sanger.ac.uk/cosmic), is one of the most comprehensive web systems describing the impact of somatic mutations in human cancer. The information is sourced directly from the published scientific literature and the Cancer Genome Project's high throughput sequencing laboratories at the Wellcome Trust Sanger Institute UK (CGP), allowing a high data quality to be maintained. The v44 release (Nov. 2009) contained the curation of 8336 papers covering 71 known cancer genes by point mutation and 30 by gene fusion. The mutation data for all these genes is being maintained up-to-date and new genes are regularly added to this list. Additionally, 4871 genes and 2764 samples have been scrutinized by the CGP laboratories. As cancer genetics moves toward the analysis of whole genomes, COSMIC is being updated to handle and display these data and increase the functionality of the website. With the rapid development of sequencing technologies, the quantity of data that can be put into COSMIC is increasing fast. We have extended our curation process to include large exome-wide candidate gene screens, recently including the first such screen from Sjoblom et al (2006), and the first TCGA screen (2008) with other large scale screening datasets being curated. In addition, whole-genome screens are becoming available with many non-coding variations. From the CGP laboratories, we already have two low-coverage genome rearrangement screens in COSMIC and this will rapidly expand, beginning with the imminent release of rearrangement scans in a set of 24 breast tumours. High-coverage genome analyses are also beginning to become available; with full genome coverage, the majority of somatic mutations in a tumour can be identified and COSMIC is being prepared to receive and display this data. With the inclusion of these new data, the COSMIC website has evolved to allow more easy access to required data. New specialization filters are in place to provide methods to search for subsets of COSMIC data in the usual user-friendly web pages. More extensive investigations can be performed using the new COSMIC Biomart, an industry-standard data mining system allowing access through web pages or programmatic interfaces. As the inclusion of new genomic data accelerates and the systems through which it can be accessed evolve, COSMIC is well placed to remain a central resource in human cancer genetics. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 93.

Genomic assessment of the evolution of the prion protein gene family in vertebrates

Prion diseases are devastating neurological disorders caused by the propagation of particles containing an alternative β−sheet-rich form of the prion protein (PrP). Genes paralogous to PrP, called Doppel and Shadoo, have been identified, that also have neuropathological relevance. To aid in the further functional characterization of PrP and its relatives, we annotated completely the PrP gene family (PrP-GF), in the genomes of 42 vertebrates, through combined strategic application of gene prediction programs and advanced remote homology detection techniques (such as HMMs, PSI-TBLASTN and pGenThreader). We have uncovered several previously undescribed paralogous genes and pseudogenes. We find that current high-quality genomic evidence indicates that the PrP relative Doppel, was likely present in the last common ancestor of present-day Tetrapoda, but was lost in the bird lineage, since its divergence from reptiles. Using the new gene annotations, we have defined the consensus of structural features that are characteristic of the PrP and Doppel structures, across diverse Tetrapoda clades. Furthermore, we describe in detail a transcribed pseudogene derived from Shadoo that is conserved across primates, and that overlaps the meiosis gene, SYCE1, thus possibly regulating its expression. In addition, we analysed the locus of PRNP/ PRND for significant conservation across the genomic DNA of eleven mammals, and determined the phylogenetic penetration of non-coding exons. The genomic evidence indicates that the second PRNP non-coding exon found in even-toed ungulates and rodents, is conserved in all high-coverage genome assemblies of primates (human, chimp, orang utan and macaque), and is, at least, likely to have fallen out of use during primate speciation. Furthermore, we have demonstrated that the PRNT gene (at the PRNP human locus) is conserved across at least sixteen mammals, and evolves like a long non-coding RNA, fashioned from fragments of ancient, long, interspersed elements. These annotations and evolutionary analyses will be of further use for functional characterisation of the PrP-GF, and will be updatable in a semi-automated fashion as more genomes accumulate.

Degeneration of Olfactory Receptor Gene Repertories in Primates: No Direct Link to Full Trichromatic Vision

Odor molecules in the environment are detected by olfactory receptors (ORs), being encoded by a large multigene family in mammalian genomes. It is generally thought that primates are vision oriented and dependent weakly on olfaction. Previous studies suggested that Old World monkeys (OWMs) and hominoids lost many functional OR genes after the divergence from New World monkeys (NWMs) due to the acquisition of well-developed trichromatic vision. To examine this hypothesis, here we analyzed OR gene repertoires of five primate species including NWMs, OWMs, and hominoids for which high-coverage genome sequences are available, together with two prosimians and tree shrews with low-coverage genomes. The results showed no significant differences in the number of functional OR genes between NWMs (marmosets) and OWMs/hominoids. Two independent analyses, identification of orthologous genes among the five primates and estimation of the numbers of ancestral genes by the reconciled tree method, did not support a sudden loss of OR genes at the branch of the OWMs/hominoids ancestor but suggested a gradual loss in every lineage. Moreover, we found that humans retain larger numbers of ancestral OR genes that were in the common ancestor of NWMs/OWMs/hominoids than orangutans and macaques and that the OR gene repertoire in humans is more similar to that of marmosets than those of orangutans and macaques. These results suggest that the degeneration of OR genes in primates cannot simply be explained by the acquisition of trichromatic vision, and our sense of smell may not be inferior to other primate species.

Spatial patterns of variation due to natural selection in humans

Empowered by technology and sampling efforts designed to facilitate genome-wide association mapping, human geneticists are now studying the geography of genetic variation in unprecedented detail. With high genomic coverage and geographic resolution, these studies are identifying loci with spatial signatures of selection, such as extreme levels of differentiation and correlations with environmental variables. Collectively, patterns at these loci are beginning to provide new insights into the process of human adaptation. Here, we review the challenges of these studies and emerging results, including how human population structure has influenced the response to novel selective pressures.

Integrated Assessment of Genomic Correlates of Protein Evolutionary Rate

Rates of evolution differ widely among proteins, but the causes and consequences of such differences remain under debate. With the advent of high-throughput functional genomics, it is now possible to rigorously assess the genomic correlates of protein evolutionary rate. However, dissecting the correlations among evolutionary rate and these genomic features remains a major challenge. Here, we use an integrated probabilistic modeling approach to study genomic correlates of protein evolutionary rate in Saccharomyces cerevisiae. We measure and rank degrees of association between (i) an approximate measure of protein evolutionary rate with high genome coverage, and (ii) a diverse list of protein properties (sequence, structural, functional, network, and phenotypic). We observe, among many statistically significant correlations, that slowly evolving proteins tend to be regulated by more transcription factors, deficient in predicted structural disorder, involved in characteristic biological functions (such as translation), biased in amino acid composition, and are generally more abundant, more essential, and enriched for interaction partners. Many of these results are in agreement with recent studies. In addition, we assess information contribution of different subsets of these protein properties in the task of predicting slowly evolving proteins. We employ a logistic regression model on binned data that is able to account for intercorrelation, non-linearity, and heterogeneity within features. Our model considers features both individually and in natural ensembles (“meta-features”) in order to assess joint information contribution and degree of contribution independence. Meta-features based on protein abundance and amino acid composition make strong, partially independent contributions to the task of predicting slowly evolving proteins; other meta-features make additional minor contributions. The combination of all meta-features yields predictions comparable to those based on paired species comparisons, and approaching the predictive limit of optimal lineage-insensitive features. Our integrated assessment framework can be readily extended to other correlational analyses at the genome scale.

The role of Havana and communities in the manual curation of unfinished vertebrate genomes

AbstractManual annotation‭ (‬the‭ "‬museum‭" ‬model of annotation‭) ‬relies on a small group of specialized curators to catalogue and classify genes according to their functional roles.‭ This‬ is both costly and time consuming and therefore is used only for model organisms with sufficient funding.‭ ‬Smaller research communities often have to rely on other models of annotation,‭ ‬mainly automated annotation‭ (‬the‭ "‬factory‭" ‬model,‭ ‬e.g.‭ ‬Ensembl‭)‬,‭ ‬and the‭ "‬jamboree‭" ‬model‭ (‬in which a group of leading biologists from the community and bioinformaticians come together for a short intensive annotation workshop‭)‬.‭ ‬At the Wellcome Trust Sanger Institute‭ (‬WTSI‭)‬,‭ ‬the Havana team provides high quality manual annotation of finished vertebrate genome sequences,‭ ‬namely human,‭ ‬mouse and zebrafish.‭ ‬We also perform the curation of specific finished regions such as the MHC in dog,‭ ‬cow and pig,‭ ‬whose whole genomes have been‭ ‬assembled from unfinished BACs or from whole genome shotgun sequences.‭ ‬In addition,‭ ‬we at Havana have also hosted annotation jamborees for the cow‭ (‬Bos taurus‭) ‬and pig‭ (‬Sus scrofa‭) ‬genomes.‭ ‬During those sessions,‭ ‬the research community had the opportunity to annotate their genes of interest under expert guidance using the custom written publicly available Otterlace annotation system,‭ ‬and the unified manual annotation guidelines.‭ ‬By making use of the tools and skills acquired during the cow and pig jamborees,‭ ‬the delegates can continue annotating their genomes remotely.‭ ‬For the pig genome,‭ ‬a highly contiguous physical map has been generated by an international effort of four laboratories (available in Pre!Ensembl) and‭ ‬is being used as a substrate for the swine genome sequencing project.‭ ‬Upcoming vertebrate genomes will be sequenced to a high depth coverage with the next generation sequencing technologies‭ (‬e.g.‭ ‬Illumina,‭ ‬454,‭ ‬SOLiD‭) ‬but will have the drawback of not being manually finished.‭ ‬Manual annotation will be more accurate than the automated predictions at coping with any assembly problems derived from these high coverage but unfinished‭ (‬or automatic pre-finished‭) ‬genomes.‭ ‬Once these inherent assembly errors are corrected and the gene structures are accurately identified with manual annotation,‭ ‬the curated genes will be incorporated and merged with the predicted gene models in Ensembl to provide a unified view of the landscape of vertebrate genomes.‭ ‬I will present an introduction to our manual annotation system and our experience using it for annotation jamborees at the WTSI.

Dynamic evolution of bitter taste receptor genes in vertebrates

BackgroundSensing bitter tastes is crucial for many animals because it can prevent them from ingesting harmful foods. This process is mainly mediated by the bitter taste receptors (T2R), which are largely expressed in the taste buds. Previous studies have identified some T2R gene repertoires, and marked variation in repertoire size has been noted among species. However, the mechanisms underlying the evolution of vertebrate T2R genes remain poorly understood.ResultsTo better understand the evolutionary pattern of these genes, we identified 16 T2R gene repertoires based on the high coverage genome sequences of vertebrates and studied the evolutionary changes in the number of T2R genes during birth-and-death evolution using the reconciled-tree method. We found that the number of T2R genes and the fraction of pseudogenes vary extensively among species. Based on the results of phylogenetic analysis, we showed that T2R gene families in teleost fishes are more diverse than those in tetrapods. In addition to the independent gene expansions in teleost fishes, frogs and mammals, lineage-specific gene duplications were also detected in lizards. Furthermore, extensive gains and losses of T2R genes were detected in each lineage during their evolution, resulting in widely differing T2R gene repertoires.ConclusionThese results further support the hypotheses that T2R gene repertoires are closely related to the dietary habits of different species and that birth-and-death evolution is associated with adaptations to dietary changes.

A genome-wide 20 K citrus microarray for gene expression analysis.

BackgroundUnderstanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genome-wide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant.ResultsWe have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a web-browsable database [1] was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability.ConclusionThis new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global studies in citrus by using it to catalogue genes expressed in citrus globular embryos.

Scanning the human genome at kilobase resolution

Normal genome variation and pathogenic genome alteration frequently affect small regions in the genome. Identifying those genomic changes remains a technical challenge. We report here the development of the DGS (Ditag Genome Scanning) technique for high-resolution analysis of genome structure. The basic features of DGS include (1) use of high-frequent restriction enzymes to fractionate the genome into small fragments; (2) collection of two tags from two ends of a given DNA fragment to form a ditag to represent the fragment; (3) application of the 454 sequencing system to reach a comprehensive ditag sequence collection; (4) determination of the genome origin of ditags by mapping to reference ditags from known genome sequences; (5) use of ditag sequences directly as the sense and antisense PCR primers to amplify the original DNA fragment. To study the relationship between ditags and genome structure, we performed a computational study by using the human genome reference sequences as a model, and analyzed the ditags experimentally collected from the well-characterized normal human DNA GM15510 and the leukemic human DNA of Kasumi-1 cells. Our studies show that DGS provides a kilobase resolution for studying genome structure with high specificity and high genome coverage. DGS can be applied to validate genome assembly, to compare genome similarity and variation in normal populations, and to identify genomic abnormality including insertion, inversion, deletion, translocation, and amplification in pathological genomes such as cancer genomes.

Survey sequencing and radiation hybrid mapping to construct comparative maps.

Radiation hybrid (RH) mapping has become one of the most well-established techniques for economically and efficiently navigating genomes of interest. The success of the technique relies on random chromosome breakage of a target genome, which is then captured by recipient cells missing a preselected marker. Selection for hybrid cells that have DNA fragments bearing the marker of choice, plus a random set of DNA fragments from the initial irradiation, generates a set of cell lines that recapitulates the genome of the target organism several-fold. Markers or genes of interest are analyzed by PCR using DNA isolated from each cell line. Statistical tools are applied to determine both the linear order of markers on each chromosome, and the confidence of each placement. The resolution of the resulting map relies on many factors, most notably the degree of breakage from the initial radiation as well as the number of hybrid clones and mean retention value.A high-resolution RH map of a genome derived from low pass or survey sequencing (coverage from 1 to 2 times) can provide essentially the same comparative data on gene order that is derived from high-coverage (greater than x7) genome sequencing. When combined with fluorescence in situ hybridization, RH maps are complete and ordered blueprints for each chromosome. They give information about the relative order and spacing of genes and markers, and allow investigators to move between target and reference genomes, such as those of mouse or human, with ease although the approach is not limited to mammal genomes.

Gene-centric characteristics of genome-wide association studies.

BackgroundThe high-throughput genotyping chips have contributed greatly to genome-wide association (GWA) studies to identify novel disease susceptibility single nucleotide polymorphisms (SNPs). The high-density chips are designed using two different SNP selection approaches, the direct gene-centric approach, and the indirect quasi-random SNPs or linkage disequilibrium (LD)-based tagSNPs approaches. Although all these approaches can provide high genome coverage and ascertain variants in genes, it is not clear to which extent these approaches could capture the common genic variants. It is also important to characterize and compare the differences between these approaches.Methodology/Principal FindingsIn our study, by using both the Phase II HapMap data and the disease variants extracted from OMIM, a gene-centric evaluation was first performed to evaluate the ability of the approaches in capturing the disease variants in Caucasian population. Then the distribution patterns of SNPs were also characterized in genic regions, evolutionarily conserved introns and nongenic regions, ontologies and pathways. The results show that, no mater which SNP selection approach is used, the current high-density SNP chips provide very high coverage in genic regions and can capture most of known common disease variants under HapMap frame. The results also show that the differences between the direct and the indirect approaches are relatively small. Both have similar SNP distribution patterns in these gene-centric characteristics.Conclusions/SignificanceThis study suggests that the indirect approaches not only have the advantage of high coverage but also are useful for studies focusing on various functional SNPs either in genes or in the conserved regions that the direct approach supports. The study and the annotation of characteristics will be helpful for designing and analyzing GWA studies that aim to identify genetic risk factors involved in common diseases, especially variants in genes and conserved regions.

Extending assembly of short DNA sequences to handle error

Inexpensive de novo genome sequencing, particularly in organisms with small genomes, is now possible using several new sequencing technologies. Some of these technologies such as that from Illumina's Solexa Sequencing, produce high genomic coverage by generating a very large number of small reads ( approximately 30 bp). While prior work shows that partial assembly can be performed by k-mer extension in error-free reads, this algorithm is unsuccessful with the sequencing error rates found in practice. We present VCAKE (Verified Consensus Assembly by K-mer Extension), a modification of simple k-mer extension that overcomes error by using high depth coverage. Though it is a simple modification of a previous approach, we show significant improvements in assembly results on simulated and experimental datasets that include error. http://152.2.15.114/~labweb/VCAKE

Reconstruction of the ancestral ferungulate karyotype by electronic chromosome painting (E-painting)

By comparing high-coverage and high-quality whole genome sequence assemblies it is now possible to reconstruct putative ancestral progenitor karyotypes, here called protokaryotypes. For this study we used the recently described electronic chromosome painting technique (E-painting) to reconstruct the karyotype of the 85 million-year-old (MYA) ferungulate ancestor. This model is primarily based on dog (Canis familiaris) and cattle (Bos taurus) genome data and is highly consistent with comparative gene mapping and chromosome painting data. The protokaryotype bears 23 autosomal chromosome pairs and the sex chromosomes and preserves most of the chromosomal associations described previously for the boreo-eutherian protokaryotype. The model indicates that five interchromosomal rearrangements occurred during the transition from the boreo-eutherian to the ferungulate ancestor. From there on 66 further interchromosomal rearrangements took place in the lineage leading to cattle and 61 further interchromosomal rearrangements in the lineage to dog.

Multiplexed Detection of Anthrax-Related Toxin Genes

Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.

Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax.

Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

Making sense of molecular signatures in the immune system.

The development of Functional Genomics technologies has opened new avenues to investigate the complexity of the immune system. Microarray technology has been particularly successful because of its relatively low cost and high genome coverage. Consequently to our ability to monitor the expression of a significant proportion of an organism genome, our understanding of the molecular dynamics behind cell differentiation and cell response has greatly improved. Molecular signatures associated to immune cells have provided important tools to investigate the molecular basis of diseases and have been often associated to diagnostic and prognostic markers. The availability of such large collection of data has stimulated the application of complex machine learning techniques in the attempt to link molecular signatures and cell physiology. Here we review the most recent developments in the analysis of molecular signatures in the immune system.