Supervised Lowess normalization of comparative genome hybridization data – application to lactococcal strain comparisons

Background: Array-based comparative genome hybridization (aCGH) is commonly used to determine the genomic content of bacterial strains. Since prokaryotes in general have less conserved genome sequences than eukaryotes, sequence divergences between the genes in the genomes used for an aCGH experiment obstruct determination of genome variations (e.g. deletions). Current normalization methods do not take into consideration sequence divergence between target and microarray features and therefore cannot distinguish a difference in signal due to systematic errors in the data or due to sequence divergence. Results: We present supervised Lowess, or S-Lowess, an application of the subset Lowess normalization method. By using a predicted subset of array features with minimal sequence divergence between the analyzed strains for the normalization procedure we remove systematic errors from dual-dye aCGH data in two steps: (1) determination of a subset of conserved genes (i.e. likely conserved genes, LCG); and (2) using the LCG for subset Lowess normalization. Subset Lowess determines the correction factors for systematic errors in the subset of array features and normalizes all array features using these correction factors. The performance of S-Lowess was assessed on aCGH experiments in which differentially labeled genomic DNA fragments of Lactococcus lactis IL1403 and L. lactis MG1363 strains were hybridized to IL1403 DNA microarrays. Since both genomes are sequenced and gene deletions identified, the success rate of different aCGH normalization methods in detecting these deletions in the MG1363 genome were determined. S-Lowess detects 97% of the deletions, whereas other aCGH normalization methods detect up to only 60% of the deletions. Conclusion: S-Lowess is implemented in a user-friendly web-tool. We demonstrate that it outperforms existing normalization methods and maximizes detection of genomic variation (e.g. deletions) from microbial aCGH data.

PreP+07: improvements of a user friendly tool to preprocess and analyse microarray data

<p>Abstract</p> <p>Background</p> <p>Nowadays, microarray gene expression analysis is a widely used technology that scientists handle but whose final interpretation usually requires the participation of a specialist. The need for this participation is due to the requirement of some background in statistics that most users lack or have a very vague notion of. Moreover, programming skills could also be essential to analyse these data. An interactive, easy to use application seems therefore necessary to help researchers to extract full information from data and analyse them in a simple, powerful and confident way.</p> <p>Results</p> <p>PreP+07 is a standalone Windows XP application that presents a friendly interface for spot filtration, inter- and intra-slide normalization, duplicate resolution, dye-swapping, error removal and statistical analyses. Additionally, it contains two unique implementation of the procedures – double scan and Supervised Lowess-, a complete set of graphical representations – MA plot, RG plot, QQ plot, PP plot, PN plot – and can deal with many data formats, such as tabulated text, GenePix GPR and ArrayPRO. PreP+07 performance has been compared with the equivalent functions in Bioconductor using a tomato chip with 13056 spots. The number of differentially expressed genes considering p-values coming from the PreP+07 and Bioconductor Limma packages were statistically identical when the data set was only normalized; however, a slight variability was appreciated when the data was both normalized and scaled.</p> <p>Conclusion</p> <p>PreP+07 implementation provides a high degree of freedom in selecting and organizing a small set of widely used data processing protocols, and can handle many data formats. Its reliability has been proven so that a laboratory researcher can afford a statistical pre-processing of his/her microarray results and obtain a list of differentially expressed genes using PreP+07 without any programming skills. All of this gives support to scientists that have been using previous PreP releases since its first version in 2003.</p

Improved analysis of bacterial CGH data beyond the log-ratio paradigm

<p>Abstract</p> <p>Background</p> <p>Existing methods for analyzing bacterial CGH data from two-color arrays are based on log-ratios only, a paradigm inherited from expression studies. We propose an alternative approach, where microarray signals are used in a different way and sequence identity is predicted using a supervised learning approach.</p> <p>Results</p> <p>A data set containing 32 hybridizations of sequenced versus sequenced genomes have been used to test and compare methods. A ROC-analysis has been performed to illustrate the ability to rank probes with respect to Present/Absent calls. Classification into Present and Absent is compared with that of a gaussian mixture model.</p> <p>Conclusion</p> <p>The results indicate our proposed method is an improvement of existing methods with respect to ranking and classification of probes, especially for multi-genome arrays.</p

Using comparative genomic hybridization to survey genomic sequence divergence across species: a proof-of-concept from Drosophila

<p>Abstract</p> <p>Background</p> <p>Genome-wide analysis of sequence divergence among species offers profound insights into the evolutionary processes that shape lineages. When full-genome sequencing is not feasible for a broad comparative study, we propose the use of array-based comparative genomic hybridization (aCGH) in order to identify orthologous genes with high sequence divergence. Here we discuss experimental design, statistical power, success rate, sources of variation and potential confounding factors. We used a spotted PCR product microarray platform from <it>Drosophila melanogaster </it>to assess sequence divergence on a gene-by-gene basis in three fully sequenced heterologous species (<it>D. sechellia</it>, <it>D. simulans</it>, and <it>D. yakuba</it>). Because complete genome assemblies are available for these species this study presents a powerful test for the use of aCGH as a tool to measure sequence divergence.</p> <p>Results</p> <p>We found a consistent and linear relationship between hybridization ratio and sequence divergence of the sample to the platform species. At higher levels of sequence divergence (< 92% sequence identity to <it>D. melanogaster</it>) ~84% of features had significantly less hybridization to the array in the heterologous species than the platform species, and thus could be identified as "diverged". At lower levels of divergence (≥ 97% identity), only 13% of genes were identified as diverged. While ~40% of the variation in hybridization ratio can be accounted for by variation in sequence identity of the heterologous sample relative to <it>D. melanogaster</it>, other individual characteristics of the DNA sequences, such as GC content, also contribute to variation in hybridization ratio, as does technical variation.</p> <p>Conclusions</p> <p>Here we demonstrate that aCGH can accurately be used as a proxy to estimate genome-wide divergence, thus providing an efficient way to evaluate how evolutionary processes and genomic architecture can shape species diversity in non-model systems. Given the increased number of species for which microarray platforms are available, comparative studies can be conducted for many interesting lineages in order to identify highly diverged genes that may be the target of natural selection.</p

Analysis of the genome content of Lactococcus garvieae by genomic interspecies microarray hybridization

BACKGROUND: Lactococcus garvieae is a bacterial pathogen that affects different animal species in addition to humans. Despite the widespread distribution and emerging clinical significance of L. garvieae in both veterinary and human medicine, there is almost a complete lack of knowledge about the genetic content of this microorganism. In the present study, the genomic content of L. garvieae CECT 4531 was analysed using bioinformatics tools and microarray-based comparative genomic hybridization (CGH) experiments. Lactococcus lactis subsp. lactis IL1403 and Streptococcus pneumoniae TIGR4 were used as reference microorganisms. RESULTS: The combination and integration of in silico analyses and in vitro CGH experiments, performed in comparison with the reference microorganisms, allowed establishment of an inter-species hybridization framework with a detection threshold based on a sequence similarity of >or= 70%. With this threshold value, 267 genes were identified as having an analogue in L. garvieae, most of which (n = 258) have been documented for the first time in this pathogen. Most of the genes are related to ribosomal, sugar metabolism or energy conversion systems. Some of the identified genes, such as als and mycA, could be involved in the pathogenesis of L. garvieae infections. CONCLUSIONS: In this study, we identified 267 genes that were potentially present in L. garvieae CECT 4531. Some of the identified genes could be involved in the pathogenesis of L. garvieae infections. These results provide the first insight into the genome content of L. garvieae.This work was supported partially by projects AGL2005-04775 and AGL2009-12447 of the Ministerio Español de Ciencia e Innovación. M. Aguado-Urda was a recipient of a grant from Centro de Vigilancia Sanitaria Veterinaria (VISAVET), and a PhD grant from the Universidad Complutense de Madrid. The work of Dr. López-Campos and Dr. Martín-Sanchez was partially funded by the COMBIOMED Network and ONTOMINEBASE reseach project (Ministerio Español de Ciencia e Innovación). The authors thank M.P. Gaya for providing the Lactococcus lactis subsp lactis IL1403 strain.S

Determining the Influence of the Extracellular Proteinase from \u3cem\u3eBrevibacterium linens\u3c/em\u3e on the Metabolism of \u3cem\u3eLactococcus lactis\u3c/em\u3e spp. \u3cem\u3elactis\u3c/em\u3e Using Functional Genomics

Since the catabolism of amino acids in cheese results in the formation of most volatile flavor compounds, a proper intracellular pool of amino acids must be established in order to produce a desirable flavor production in cheese. Generation of this pool of amino acids requires complex interactions among casein and its derivatives, proteolytic enzymes, and transport systems in the associated bacteria, including lactococci. In this project, we hypothesized that casein hydrolysis by the extracellular proteinases of Brevibacterium linens BL2 modulates the expression profile of proteolytic related genes in Lactococcus lactis spp. lactis IL1403. In order to monitor the global gene regulation patterns in L. lactis ssp. lactis IL1403, a high-throughput gene expression tool was needed to study the gene expression profiles on a genomic scale. In this project, we developed a novel oligonucleotide-based filter DNA array protocol for this purpose. The success of this oligonucleotide-based DNA array was dependent on technical innovations including polyI tailing, indirect high density biotin labeling, careful probe design, and integrated computational data analysis. The utility and validity of this protocol were demonstrated by profiling the expression of 375 metabolically related genes in L. lactis ssp. lactis IL1403 during heat, acid, and osmotic stresses. Subsequently the DNA macroarray was used to profile the gene expression changes of L. lactis spp. lactis IL1403 growing in a peptide-limited medium, in a casitone-based peptide-rich medium, and in a casein hydrolyte by B. linens BL2 proteolytic enzymes. L. lactis ssp. lactis IL1403 experienced nitrogen starvation even with an abundance of peptide resources because of lack of expression of peptide transporter genes. Conversely, a peptide pool generated by B. linens BL2 proteolytic activities was sufficient to sustain the growth of L. lactis ssp. lactis IL1403. The repression of the peptide transporter and other peptidase genes of L. lactis ssp. lactis IL1403 was relieved in this medium. Interestingly, the Opt system, a di-tripeptide transporter, was used as a primary peptide transporter, instead of the Opp system whose genes were not actively transcripted in IL1403. We also conducted additional experiments to further describe the protease in B. linens BL2 responsible for the peptide pool generation. This enzyme was secreted as a non-active zymogen and matured into the active protease. Both proteolysis and maturation processes were regulated. Collectively, this work demonstrated that a unique protease of B. linens BL2 generated a pool of pep tides transportable by L. lactis IL1403 and induced changes in gene expression in L. lactis IL1403. Consequently, this body of work demonstrated the hypothesis to be true

Sensitivity of Lactococcus garvieae isolated from rainbow trout to some Iranian medicinal herbs

Lactococcus garvieae is one of the most important bacterial pathogens that affect different farmed fish species in many countries. Some of the medicinal plants and their extracts have an antibacterial activity. In this study, antibacterial activity of ethanol extracts and essential oils of fifteen Iranian medicinal plants including, Satureja bachtiarica Bunge., Thymus daenensis Celak., Stachys lavandulifolia Vahl., Zataria multifora Boiss., Thymbra spicata L., Teucrium polium L. (Lamiaceae); Myrtus communis L. (Myrtaceae); Heracleum lasiopetalum Boiss., Kelussia odoratissima Mozaff., Bunium persicum (Boiss.) K.-Pol., Echiophora platyloba L. (Apiaceae); Punica granatum L. (Punicaceae); Qurcus branti Lindle (Fagaceae); Glycyrrhiza glabra L., Alhagi maurorum L. (Fabaceae) were investigated against Lactococcus garvieae isolated from rainbow trout (Oncorhynchus mykiss) by agar disc diffusion and serial dilution assays. Just the extracts of M. communis and T. daenensis showed relatively antibacterial activity against L. garvieae. Almost the essential oils showed strong antibacterial activity against fish pathogen. The results obtained appeared to confirm the antibacterial potential of the essential oils of B. persicum, Z. multifora, T. daenensis, S. bachtiarica and T. spicata against L. garvieae isolated from rainbow trout. The MICs and MLCs of Z. multifora essential oil against L. garvieae were found as 4-8 µl/ml

Gene duplication in an African cichlid adaptive radiation

BACKGROUND: Gene duplication is a source of evolutionary innovation and can contribute to the divergence of lineages; however, the relative importance of this process remains to be determined. The explosive divergence of the African cichlid adaptive radiations provides both a model for studying the general role of gene duplication in the divergence of lineages and also an exciting foray into the identification of genomic features that underlie the dramatic phenotypic and ecological diversification in this particular lineage. We present the first genome-wide study of gene duplication in African cichlid fishes, identifying gene duplicates in three species belonging to the Lake Malawi adaptive radiation (Metriaclima estherae, Protomelas similis, Rhamphochromis “chilingali”) and one closely related species from a non-radiated riverine lineage (Astatotilapia tweddlei). RESULTS: Using Astatotilapia burtoni as reference, microarray comparative genomic hybridization analysis of 5689 genes reveals 134 duplicated genes among the four cichlid species tested. Between 51 and 55 genes were identified as duplicated in each of the three species from the Lake Malawi radiation, representing a 38%–49% increase in number of duplicated genes relative to the non-radiated lineage (37 genes). Duplicated genes include several that are involved in immune response, ATP metabolism and detoxification. CONCLUSIONS: These results contribute to our understanding of the abundance and type of gene duplicates present in cichlid fish lineages. The duplicated genes identified in this study provide candidates for the analysis of functional relevance with regard to phenotype and divergence. Comparative sequence analysis of gene duplicates can address the role of positive selection and adaptive evolution by gene duplication, while further study across the phylogenetic range of cichlid radiations (and more generally in other adaptive radiations) will determine whether the patterns of gene duplication seen in this study consistently accompany rapid radiation