Mayday - integrative analytics for expression data

<p>Abstract</p> <p>Background</p> <p>DNA Microarrays have become the standard method for large scale analyses of gene expression and epigenomics. The increasing complexity and inherent noisiness of the generated data makes visual data exploration ever more important. Fast deployment of new methods as well as a combination of predefined, easy to apply methods with programmer's access to the data are important requirements for any analysis framework. Mayday is an open source platform with emphasis on visual data exploration and analysis. Many built-in methods for clustering, machine learning and classification are provided for dissecting complex datasets. Plugins can easily be written to extend Mayday's functionality in a large number of ways. As Java program, Mayday is platform-independent and can be used as Java WebStart application without any installation. Mayday can import data from several file formats, database connectivity is included for efficient data organization. Numerous interactive visualization tools, including box plots, profile plots, principal component plots and a heatmap are available, can be enhanced with metadata and exported as publication quality vector files.</p> <p>Results</p> <p>We have rewritten large parts of Mayday's core to make it more efficient and ready for future developments. Among the large number of new plugins are an automated processing framework, dynamic filtering, new and efficient clustering methods, a machine learning module and database connectivity. Extensive manual data analysis can be done using an inbuilt R terminal and an integrated SQL querying interface. Our visualization framework has become more powerful, new plot types have been added and existing plots improved.</p> <p>Conclusions</p> <p>We present a major extension of Mayday, a very versatile open-source framework for efficient micro array data analysis designed for biologists and bioinformaticians. Most everyday tasks are already covered. The large number of available plugins as well as the extension possibilities using compiled plugins and ad-hoc scripting allow for the rapid adaption of Mayday also to very specialized data exploration. Mayday is available at <url>http://microarray-analysis.org</url>.</p

iHAT: interactive Hierarchical Aggregation Table for Genetic Association Data

In the search for single-nucleotide polymorphisms which influence the observable phenotype, genome wide association studies have become an important technique for the identification of associations between genotype and phenotype of a diverse set of sequence-based data. We present a methodology for the visual assessment of single-nucleotide polymorphisms using interactive hierarchical aggregation techniques combined with methods known from traditional sequence browsers and cluster heatmaps. Our tool, the interactive Hierarchical Aggregation Table (iHAT), facilitates the visualization of multiple sequence alignments, associated metadata, and hierarchical clusterings. Different color maps and aggregation strategies as well as filtering options support the user in finding correlations between sequences and metadata. Similar to other visualizations such as parallel coordinates or heatmaps, iHAT relies on the human pattern-recognition ability for spotting patterns that might indicate correlation or anticorrelation. We demonstrate iHAT using artificial and real-world datasets for DNA and protein association studies as well as expression Quantitative Trait Locus data

An eQTL biological data visualization challenge and approaches from the visualization community

In 2011, the IEEE VisWeek conferences inaugurated a symposium on Biological Data Visualization. Like other domain-oriented Vis symposia, this symposium's purpose was to explore the unique characteristics and requirements of visualization within the domain, and to enhance both the Visualization and Bio/Life-Sciences communities by pushing Biological data sets and domain understanding into the Visualization community, and well-informed Visualization solutions back to the Biological community. Amongst several other activities, the BioVis symposium created a data analysis and visualization contest. Unlike many contests in other venues, where the purpose is primarily to allow entrants to demonstrate tour-de-force programming skills on sample problems with known solutions, the BioVis contest was intended to whet the participants' appetites for a tremendously challenging biological domain, and simultaneously produce viable tools for a biological grand challenge domain with no extant solutions. For this purpose expression Quantitative Trait Locus (eQTL) data analysis was selected. In the BioVis 2011 contest, we provided contestants with a synthetic eQTL data set containing real biological variation, as well as a spiked-in gene expression interaction network influenced by single nucleotide polymorphism (SNP) DNA variation and a hypothetical disease model. Contestants were asked to elucidate the pattern of SNPs and interactions that predicted an individual's disease state. 9 teams competed in the contest using a mixture of methods, some analytical and others through visual exploratory methods. Independent panels of visualization and biological experts judged entries. Awards were given for each panel's favorite entry, and an overall best entry agreed upon by both panels. Three special mention awards were given for particularly innovative and useful aspects of those entries. And further recognition was given to entries that correctly answered a bonus question about how a proposed "gene therapy" change to a SNP might change an individual's disease status, which served as a calibration for each approaches' applicability to a typical domain question. In the future, BioVis will continue the data analysis and visualization contest, maintaining the philosophy of providing new challenging questions in open-ended and dramatically underserved Bio/Life Sciences domains

Mayday SeaSight: Combined Analysis of Deep Sequencing and Microarray Data

Recently emerged deep sequencing technologies offer new high-throughput methods to quantify gene expression, epigenetic modifications and DNA-protein binding. From a computational point of view, the data is very different from that produced by the already established microarray technology, providing a new perspective on the samples under study and complementing microarray gene expression data. Software offering the integrated analysis of data from different technologies is of growing importance as new data emerge in systems biology studies. Mayday is an extensible platform for visual data exploration and interactive analysis and provides many methods for dissecting complex transcriptome datasets. We present Mayday SeaSight, an extension that allows to integrate data from different platforms such as deep sequencing and microarrays. It offers methods for computing expression values from mapped reads and raw microarray data, background correction and normalization and linking microarray probes to genomic coordinates. It is now possible to use Mayday's wealth of methods to analyze sequencing data and to combine data from different technologies in one analysis

A New Panel-Based Next-Generation Sequencing Method for ADME Genes Reveals Novel Associations of Common and Rare Variants With Expression in a Human Liver Cohort

We developed a panel-based NGS pipeline for comprehensive analysis of 340 genes involved in absorption, distribution, metabolism and excretion (ADME) of drugs, other xenobiotics, and endogenous substances. The 340 genes comprised phase I and II enzymes, drug transporters and regulator/modifier genes within their entire coding regions, adjacent intron regions and 5′ and 3′UTR regions, resulting in a total panel size of 1,382 kbp. We applied the ADME NGS panel to sequence genomic DNA from 150 Caucasian liver donors with available comprehensive gene expression data. This revealed an average read-depth of 343 (range 27–811), while 99% of the 340 genes were covered on average at least 100-fold. Direct comparison of variant annotation with 363 available genotypes determined independently by other methods revealed an overall accuracy of &gt;99%. Of 15,727 SNV and small INDEL variants, 12,022 had a minor allele frequency (MAF) below 2%, including 8,937 singletons. In total we found 7,273 novel variants. Functional predictions were computed for coding variants (n = 4,017) by three algorithms (Polyphen 2, Provean, and SIFT), resulting in 1,466 variants (36.5%) concordantly predicted to be damaging, while 1,019 variants (25.4%) were predicted to be tolerable. In agreement with other studies we found that less common variants were enriched for deleterious variants. Cis-eQTL analysis of variants with (MAF ≥ 2%) revealed significant associations for 90 variants in 31 genes after Bonferroni correction, most of which were located in non-coding regions. For less common variants (MAF &lt; 2%), we applied the SKAT-O test and identified significant associations to gene expression for ADH1C and GSTO1. Moreover, our data allow comparison of functional predictions with additional phenotypic data to prioritize variants for further analysis

Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease-Towards a Point-of-Care Test

Background As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP),a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents. Methodology/Principal Findings Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR),IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%;100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%;cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays. Conclusions/Significance Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level

O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis

Background: Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. Methods: A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Results: Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. Conclusions: The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis

The dynamic architecture of the metabolic switch in Streptomyces coelicolor

[EN] Background: During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples.Results: Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.Conclusions: Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological settingSIWe are very grateful to Mervyn Bibb for his generous support with the Affymetrix custom microarray design. We acknowledge the excellent technical help of K. Klein, S. Poths, M. Walter, A. Øverby and E. Hansen. This project was supported by grants of the ERA-NET SySMO Project [GEN2006-27745-E/SYS]: (P-UK-01-11-3i) and the Research Council of Norway [project no. 181840/I30

Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines : a case report

Abstract Background Cancer vaccines can effectively establish clinically relevant tumor immunity. Novel sequencing approaches rapidly identify the mutational fingerprint of tumors, thus allowing to generate personalized tumor vaccines within a few weeks from diagnosis. Here, we report the case of a 62-year-old patient receiving a four-peptide-vaccine targeting the two sole mutations of his pancreatic tumor, identified via exome sequencing. Methods Vaccination started during chemotherapy in second complete remission and continued monthly thereafter. We tracked IFN-γ+ T cell responses against vaccine peptides in peripheral blood after 12, 17 and 34 vaccinations by analyzing T-cell receptor (TCR) repertoire diversity and epitope-binding regions of peptide-reactive T-cell lines and clones. By restricting analysis to sorted IFN-γ-producing T cells we could assure epitope-specificity, functionality, and TH1 polarization. Results A peptide-specific T-cell response against three of the four vaccine peptides could be detected sequentially. Molecular TCR analysis revealed a broad vaccine-reactive TCR repertoire with clones of discernible specificity. Four identical or convergent TCR sequences could be identified at more than one time-point, indicating timely persistence of vaccine-reactive T cells. One dominant TCR expressing a dual TCRVα chain could be found in three T-cell clones. The observed T-cell responses possibly contributed to clinical outcome: The patient is alive 6 years after initial diagnosis and in complete remission for 4 years now. Conclusions Therapeutic vaccination with a neoantigen-derived four-peptide vaccine resulted in a diverse and long-lasting immune response against these targets which was associated with prolonged clinical remission. These data warrant confirmation in a larger proof-of concept clinical trial