ABSTRACT
Glucolipids (GLs) are glycolipid biosurfactants with promising properties. These GLs are composed of glucose attached to a hydroxy fatty acid through a ω and/or ω-1 glycosidic linkage. Up until today these interesting molecules could only be produced using an engineered Starmerella bombicola strain (∆ugtB1::URA3 G9) producing GLs instead of sophorolipids, albeit with a very low average productivity (0.01 g·L-1 ·h-1 ). In this study, we investigated the reason(s) for this via reverse-transcription quantitative polymerase chain reaction and Liquid chromatography-multireaction monitoring-mass spectrometry. We found that all glycolipid biosynthetic genes and enzymes were downregulated in the ∆ugtB1 G9 strain in comparison to the wild type. The underlying reason for this downregulation was further investigated by performing quantitative metabolome comparison of the ∆ugtB1 G9 strain with the wild type and two other engineered strains also tinkered in their glycolipid biosynthetic gene cluster. This analysis revealed a clear distortion of the entire metabolism of the ∆ugtB1 G9 strain compared to all the other strains. Because the parental strain of the former was a spontaneous ∆ura3 mutant potentially containing other "hidden" mutations, a new GL production strain was generated based on a rationally engineered ∆ura3 mutant (PT36). Indeed, a 50-fold GL productivity increase (0.51 g·L-1 ·h-1 ) was obtained with the new ∆ugtB1::URA3 PT36 strain compared with the G9-based strain (0.01 g·L-1 ·h-1 ) in a 10 L bioreactor experiment, yielding 118 g/L GLs instead of 8.39 g/L. Purification was investigated and basic properties of the purified GLs were determined. This study forms the base for further development and optimization of S. bombicola as a production platform strain for (new) biochemicals.
Subject(s)
Glycolipids , Metabolic Engineering/methods , Saccharomycetales , Surface-Active Agents , Bioreactors , Fermentation , Glycolipids/chemistry , Glycolipids/genetics , Glycolipids/metabolism , Metabolome/genetics , Saccharomycetales/genetics , Saccharomycetales/metabolism , Surface-Active Agents/chemistry , Surface-Active Agents/metabolismABSTRACT
BACKGROUND: Although the sequencing landscape is rapidly evolving and sequencing costs are continuously decreasing, whole genome sequencing is still too expensive for use on a routine basis. Targeted resequencing of only the regions of interest decreases both costs and the complexity of the downstream data-analysis. Various target enrichment strategies are available, but none of them obtain the degree of coverage uniformity, flexibility and specificity of PCR-based enrichment. On the other hand, the biggest limitation of target enrichment by PCR is the need to design large numbers of partially overlapping assays to cover the target. RESULTS: To overcome the aforementioned hurdles, we have developed primerXL, a state-of-the-art PCR primer design pipeline for targeted resequencing. It uses an optimized design criteria relaxation cascade and a thorough downstream in silico evaluation process to generate high quality singleplex PCR assays, reducing the need for amplicon normalization, and outperforming other target enrichment strategies and similar primer design tools when considering assay quality, coverage uniformity and target coverage. Results of four different sequencing projects with 2348 amplicons in total covering 470 kb are presented. PrimerXL can be accessed at www.primerxl.org . CONCLUSION: PrimerXL is an state-of-the-art, easy to use primer design webtool capable of generating high-quality targeted resequencing assays. The workflow is fully customizable to suit every researchers' needs, while an innovative relaxation cascade ensures maximal target coverage.
Subject(s)
DNA Primers/metabolism , High-Throughput Nucleotide Sequencing/methods , Polymerase Chain Reaction/methods , User-Computer Interface , Animals , DNA Primers/genetics , Humans , Plants/genetics , Plants/metabolism , Polymorphism, Single Nucleotide , Sequence Analysis, DNAABSTRACT
MYCN is a master regulator controlling many processes necessary for tumor cell survival. Here, we unravel a microRNA network that causes tumor suppressive effects in MYCN-amplified neuroblastoma cells. In profiling studies, histone deacetylase (HDAC) inhibitor treatment most strongly induced miR-183. Enforced miR-183 expression triggered apoptosis, and inhibited anchorage-independent colony formation in vitro and xenograft growth in mice. Furthermore, the mechanism of miR-183 induction was found to contribute to the cell death phenotype induced by HDAC inhibitors. Experiments to identify the HDAC(s) involved in miR-183 transcriptional regulation showed that HDAC2 depletion induced miR-183. HDAC2 overexpression reduced miR-183 levels and counteracted the induction caused by HDAC2 depletion or HDAC inhibitor treatment. MYCN was found to recruit HDAC2 in the same complexes to the miR-183 promoter, and HDAC2 depletion enhanced promoter-associated histone H4 pan-acetylation, suggesting epigenetic changes preceded transcriptional activation. These data reveal miR-183 tumor suppressive properties in neuroblastoma that are jointly repressed by MYCN and HDAC2, and suggest a novel way to bypass MYCN function.
Subject(s)
Histone Deacetylase 2/metabolism , MicroRNAs/metabolism , Neuroblastoma/genetics , Nuclear Proteins/metabolism , Oncogene Proteins/metabolism , Animals , Cell Death , Cell Line, Tumor , Histone Deacetylase Inhibitors/pharmacology , Humans , Mice , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , N-Myc Proto-Oncogene Protein , Neuroblastoma/metabolism , Neuroblastoma/pathology , Promoter Regions, Genetic , Signal TransductionABSTRACT
BACKGROUND: Genome-sequencing studies have led to an immense increase in the number of known single-nucleotide polymorphisms (SNPs). Designing primers that anneal to regions devoid of SNPs has therefore become challenging. We studied the impact of one or more mismatches in primer-annealing sites on different quantitative PCR (qPCR)-related parameters, such as quantitative cycle (Cq), amplification efficiency, and reproducibility. METHODS: We used synthetic templates and primers to assess the effect of mismatches at primer-annealing sites on qPCR assay performance. Reactions were performed with 5 commercially available master mixes. We studied the effects of the number, type, and position of priming mismatches on Cq value, PCR efficiency, reproducibility, and yield. RESULTS: The impact of mismatches was most pronounced for the number of mismatched nucleotides and for their distance from the 3' end of the primer. In addition, having ≥4 mismatches in a single primer or having 3 mismatches in one primer and 2 in the other was required to block a reaction completely. Finally, the degree of the mismatch effect was concentration independent for single mismatches, whereas concentration independence failed at higher template concentrations as the number of mismatches increased. CONCLUSIONS: Single mismatches located >5 bp from the 3' end have a moderate effect on qPCR amplification and can be tolerated. This finding, together with the concentration independence for single mismatches and the complete blocking of the PCR reaction for ≥4 mismatches, can help to chart mismatch behavior in qPCR reactions and increase the rate of successful primer design for sequences with a high SNP density or for homologous regions of sequence.
Subject(s)
Base Pair Mismatch , DNA Primers/genetics , Polymerase Chain Reaction/methods , Polymorphism, Single Nucleotide , Reproducibility of ResultsABSTRACT
While a growing body of evidence implicates regulatory miRNA modules in various aspects of human disease and development, insights into specific miRNA function remain limited. Here, we present an innovative approach to elucidate tissue-specific miRNA functions that goes beyond miRNA target prediction and expression correlation. This approach is based on a multi-level integration of corresponding miRNA and mRNA gene expression levels, miRNA target prediction, transcription factor target prediction and mechanistic models of gene network regulation. Predicted miRNA functions were either validated experimentally or compared to published data. The predicted miRNA functions are accessible in the miRNA bodymap, an interactive online compendium and mining tool of high-dimensional newly generated and published miRNA expression profiles. The miRNA bodymap enables prioritization of candidate miRNAs based on their expression pattern or functional annotation across tissue or disease subgroup. The miRNA bodymap project provides users with a single one-stop data-mining solution and has great potential to become a community resource.
Subject(s)
MicroRNAs/metabolism , Software , Animals , Cell Line, Tumor , Data Mining , Gene Expression Profiling , Gene Expression Regulation , Gene Regulatory Networks , Genomics , Humans , Mice , Models, Genetic , Molecular Sequence Annotation , RNA, Messenger/metabolism , Rats , Transcription Factors/metabolismABSTRACT
Neuroblastoma (NB) is a paediatric tumour with a remarkable diverse clinical behaviour. Approximately half of the high stage aggressive tumours are characterized by MYCN gene amplification but our understanding of the role of MYCN in NB oncogenesis is incomplete. Previous studies have shown that MYCN expression is inversely correlated with expression of Dickkopf-3 (DKK3), a gene encoding an extracellular protein with presumed tumour suppressor activity, but direct MYCN regulation of DKK3 was excluded leaving the mechanism of regulation unexplained. Given the recently established role of MYCN-regulated miRNAs in downregulation of protein-coding genes and predicted seeds for miR-17-92 cluster members within the DKK3 3'UTR, we hypothesized that this mechanism would act in MYCN regulation of DKK3. To investigate this, we used a validated miR-17-92-inducible cellular system and could demonstrate robust downregulation of DKK3 mRNA and protein levels upon miR-17-92 overexpression. Next, two of the three predicted miRNAs, miR-19b and miR-92a, were shown to lower DKK3 protein levels, in addition to measurable DKK3 mRNA knock-down by miR-92a. Direct interaction between miR-19b or miR-92a and the 3'UTR of DKK3 was validated using luciferase reporter assays. In conclusion, this study demonstrates that the MYCN-induced downregulation of DKK3 results from direct upregulation of miR-17-92 components effecting both DKK3 mRNA stability and translation which further contributes to the pleiotropic oncogenic effect of elevated MYCN levels. The strict MYCN-mediated regulation of DKK3 is suggestive for an important downstream function of the MYCN protein and thus warrants further investigations to unravel the role of DKK3 in NB.
Subject(s)
Intercellular Signaling Peptides and Proteins/physiology , MicroRNAs/physiology , Neuroblastoma/genetics , Nuclear Proteins/physiology , Oncogene Proteins/physiology , 3' Untranslated Regions , Adaptor Proteins, Signal Transducing , Cell Line, Tumor , Chemokines , Humans , Intercellular Signaling Peptides and Proteins/genetics , MicroRNAs/analysis , N-Myc Proto-Oncogene Protein , Neuroblastoma/etiology , RNA, Long Noncoding , Up-RegulationABSTRACT
Neuroblastoma is an aggressive embryonal tumor that accounts for â¼15% of childhood cancer deaths. Hitherto, despite the availability of comprehensive genomic data on DNA copy number changes in neuroblastoma, relatively little is known about the genes driving neuroblastoma tumorigenesis. In this study, high resolution array comparative genome hybridization (CGH) was performed on 188 primary neuroblastoma tumors and 33 neuroblastoma cell lines to search for previously undetected recurrent DNA copy number gains and losses. A new recurrent distal chromosome 1q deletion (del(1)(q42.2qter)) was detected in seven cases. Further analysis of available array CGH datasets revealed 13 additional similar distal 1q deletions. The majority of all detected 1q deletions was found in high risk 11q deleted tumors without MYCN amplification (Fisher exact test p = 5.61 × 10(-5) ). Using ultra-high resolution (â¼115 bp resolution) custom arrays covering the breakpoints on 1q for 11 samples, clustering of nine breakpoints was observed within a 12.5-kb region, of which eight were found in a 7-kb copy number variable region, whereas the remaining two breakpoints were colocated 1.4-Mb proximal. The commonly deleted region contains one miRNA (hsa-mir-1537), four transcribed ultra conserved region elements (uc.43-uc.46) and 130 protein coding genes including at least two bona fide tumor suppressor genes, EGLN1 (or PHD2) and FH. This finding further contributes to the delineation of the genomic profile of aggressive neuroblastoma, offers perspectives for the identification of genes contributing to the disease phenotype and may be relevant in the light of assessment of response to new molecular treatments.
Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 11 , Gene Dosage , Neuroblastoma/genetics , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Cell Line, Tumor , Comparative Genomic Hybridization , Fumarate Hydratase/genetics , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases , N-Myc Proto-Oncogene Protein , Procollagen-Proline Dioxygenase/geneticsABSTRACT
PURPOSE: Leber congenital amaurosis (LCA) is a rare congenital retinal dystrophy associated with 16 genes. Recent breakthroughs in LCA gene therapy offer the first prospect of treating inherited blindness, which requires an unequivocal and early molecular diagnosis. While present genetic tests do not address this due to a tremendous genetic heterogeneity, massively parallel sequencing (MPS) strategies might bring a solution. Here, we developed a comprehensive molecular test for LCA based on targeted MPS of all exons of 16 known LCA genes. METHODS: We designed a unique and flexible workflow for targeted resequencing of all 236 exons from 16 LCA genes based on quantitative PCR (qPCR) amplicon ligation, shearing, and parallel sequencing of multiple patients on a single lane of a short-read sequencer. Twenty-two prescreened LCA patients were included, five of whom had a known molecular cause. RESULTS: Validation of 107 variations was performed as proof of concept. In addition, the causal genetic defect and a single heterozygous mutation were identified in 3 and 5, respectively, of 17 patients without previously identified mutations. CONCLUSION: We propose a novel targeted MPS-based approach that is suitable for accurate, fast, and cost-effective early molecular testing in LCA, and easily applicable in other genetic disorders.
Subject(s)
High-Throughput Nucleotide Sequencing/methods , Leber Congenital Amaurosis/diagnosis , Molecular Diagnostic Techniques/methods , Adaptor Proteins, Signal Transducing , Antigens, Neoplasm/genetics , Biomarkers/analysis , Blindness/congenital , Blindness/genetics , Carrier Proteins/genetics , Case-Control Studies , Cell Cycle Proteins , Child , Child, Preschool , Consanguinity , Cytoskeletal Proteins , Exons/genetics , Eye Proteins/genetics , Genetic Heterogeneity , Guanylate Cyclase/genetics , Heterozygote , Homeodomain Proteins/genetics , Humans , Leber Congenital Amaurosis/genetics , Membrane Proteins/genetics , Mutation , Neoplasm Proteins/genetics , Nerve Tissue Proteins/genetics , Real-Time Polymerase Chain Reaction/methods , Receptors, Cell Surface/genetics , Trans-Activators/genetics , Validation Studies as Topic , cis-trans-Isomerases/geneticsABSTRACT
To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs. Apart from three larger distant deletions, a de novo deletion as small as 7.4 kb was found at 283 kb 5' to FOXL2. The deletion appeared to be triggered by an H-DNA-induced double-stranded break (DSB). In addition, it disrupts a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs. The regulatory potential of the deleted CNCs was substantiated by in vitro luciferase assays. Interestingly, Chromosome Conformation Capture (3C) of a 625 kb region surrounding FOXL2 in expressing cellular systems revealed physical interactions of three upstream fragments and the FOXL2 core promoter. Importantly, one of these contains the 7.4 kb deleted fragment. Overall, this study revealed the smallest distant deletion causing monogenic disease and impacts upon the concept of mutation screening in human disease and developmental disorders in particular.
Subject(s)
5' Untranslated Regions , Blepharophimosis/genetics , Forkhead Transcription Factors/genetics , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Sequence Deletion , Cell Line , Conserved Sequence , DNA Mutational Analysis , Forkhead Box Protein L2 , Humans , Protein BindingABSTRACT
RTPrimerDB (http://www.rtprimerdb.org) is a freely accessible database and analysis tool for real-time quantitative PCR assays. RTPrimerDB includes records with user submitted assays that are linked to genome information from reference databases and quality controlled using an in silico assay evaluation system. The primer evaluation tools intended to assess the specificity and to detect features that could negatively affect the amplification efficiency are combined into a pipeline to test custom-designed primer and probe sequences. An improved user feedback system guides users and submitters to enter practical remarks and details about experimental evaluation analyses. The database is linked with reference databases to allow the submission of assays for all genes and organisms officially registered in Entrez Gene and RefSeq. Records in RTPrimerDB are assigned unique and stable identifiers. The content is provided via an interactive web-based search system and is available for download in the recently developed RDML format and as bulk export file. RTPrimerDB is a one-stop portal for high-quality and highly annotated real-time PCR assays.
Subject(s)
DNA Primers/chemistry , Databases, Nucleic Acid , Oligonucleotide Probes/chemistry , Polymerase Chain Reaction , User-Computer InterfaceABSTRACT
The XML-based Real-Time PCR Data Markup Language (RDML) has been developed by the RDML consortium (http://www.rdml.org) to enable straightforward exchange of qPCR data and related information between qPCR instruments and third party data analysis software, between colleagues and collaborators and between experimenters and journals or public repositories. We here also propose data related guidelines as a subset of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to guarantee inclusion of key data information when reporting experimental results.
Subject(s)
Guidelines as Topic , Polymerase Chain Reaction/standards , Software , Terminology as Topic , InternetABSTRACT
The quantitative polymerase chain reaction (qPCR) is widely utilized for gene expression analysis. However, the lack of robust strategies for cross laboratory data comparison hinders the ability to collaborate or perform large multicentre studies conducted at different sites. In this study we introduced and validated a workflow that employs universally applicable, quantifiable external oligonucleotide standards to address this question. Using the proposed standards and data-analysis procedure, we obtained a perfect concordance between expression values from eight different genes in 366 patient samples measured on three different qPCR instruments and matching software, reagents, plates and seals, demonstrating the power of this strategy to detect and correct inter-run variation and to enable exchange of data between different laboratories, even when not using the same qPCR platform.
Subject(s)
DNA Primers/standards , Gene Expression , Reverse Transcriptase Polymerase Chain Reaction/standards , Calibration , HumansABSTRACT
BACKGROUND: Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions. RESULTS: We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data. CONCLUSIONS: We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available.
Subject(s)
Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods , Software , Base Sequence , Databases, Genetic , Molecular Sequence Data , Sequence Alignment , User-Computer InterfaceABSTRACT
PURPOSE: Neuroblastoma is a heterogeneous childhood tumor with poor survival outcome for the aggressive type despite intensive multimodal therapies. In this study, we aimed to identify new treatment options for neuroblastoma based on integrative genomic analysis. EXPERIMENTAL DESIGN: The Connectivity Map is a database comprising expression profiles in response to known therapeutic compounds. This renders it a useful tool in the search for potential therapeutic compounds based on comparison of gene expression profiles of diseased cells and a database of profiles in response to known therapeutic compounds. We have used this strategy in the search for new therapeutic molecules for neuroblastoma based on data of an integrative meta-analysis of gene copy number and expression profiles from 146 primary neuroblastoma tumors and normal fetal neuroblasts. RESULTS: In a first step, a 132-gene classifier was established that discriminates three major genomic neuroblastoma subgroups, reflecting inherent differences in gene expression between these subgroups. Subsequently, we screened the Connectivity Map database using gene lists generated by comparing expression profiles of fetal adrenal neuroblasts and the genomic subgroups of neuroblastomas. A putative therapeutic effect was predicted for several compounds of which six were empirically tested. A significant reduction in cell viability was shown for five of these molecules: 17-allylamino-geldanamycin, monorden, fluphenazine, trichostatin, and rapamycin. CONCLUSIONS: This proof-of-principle study indicates that an integrative genomic meta-analysis approach with inclusion of neuroblast data enables the identification of promising compounds for treatment of children with neuroblastoma. Further studies are warranted to explore in detail the therapeutic potential of these compounds.
Subject(s)
Antineoplastic Agents/pharmacology , Gene Expression Profiling/statistics & numerical data , Meta-Analysis as Topic , Neuroblastoma/genetics , Cell Line, Tumor , Cell Survival/drug effects , Gene Expression Profiling/classification , Genomics/methods , Humans , Neuroblastoma/classification , Neuroblastoma/pathology , Neurons/metabolismABSTRACT
For a wide range of diseases, SNPs in the genome are the underlying mechanism of dysfunction. Therefore, targeted detection of these variations is of high importance for early diagnosis and (familial) screenings. While allele-specific PCR has been around for many years, its adoption for SNP genotyping or somatic mutation detection has been hampered by its low discriminating power and high costs. To tackle this, we developed a cost-effective qPCR based method, able to detect SNPs in a robust and specific manner. This study describes how to combine the basic principles of allele-specific PCR (the combination of a wild type and variant primer) with the straightforward readout of DNA-binding dye based qPCR technology. To enhance the robustness and discriminating power, an artificial mismatch in the allele-specific primer was introduced. The resulting method, called double-mismatch allele-specific qPCR (DMAS-qPCR), was successfully validated using 12 SNPs and 15 clinically relevant somatic mutations on 48 cancer cell lines. It is easy to use, does not require labeled probes and is characterized by high analytical sensitivity and specificity. DMAS-qPCR comes with a complimentary online assay design tool, available for the whole scientific community, enabling researchers to design custom assays and implement those as a diagnostic test.
Subject(s)
Cost-Benefit Analysis , Genetic Diseases, Inborn/diagnosis , Genotyping Techniques/economics , Genotyping Techniques/methods , Real-Time Polymerase Chain Reaction/economics , Real-Time Polymerase Chain Reaction/methods , Alleles , Genotype , Humans , Polymorphism, Single Nucleotide , Sensitivity and SpecificityABSTRACT
BACKGROUND: The use of linked data in the Semantic Web is a promising approach to add value to nutrition research. An ontology, which defines the logical relationships between well-defined taxonomic terms, enables linking and harmonizing research output. To enable the description of domain-specific output in nutritional epidemiology, we propose the Ontology for Nutritional Epidemiology (ONE) according to authoritative guidance for nutritional epidemiology. METHODS: Firstly, a scoping review was conducted to identify existing ontology terms for reuse in ONE. Secondly, existing data standards and reporting guidelines for nutritional epidemiology were converted into an ontology. The terms used in the standards were summarized and listed separately in a taxonomic hierarchy. Thirdly, the ontologies of the nutritional epidemiologic standards, reporting guidelines, and the core concepts were gathered in ONE. Three case studies were included to illustrate potential applications: (i) annotation of existing manuscripts and data, (ii) ontology-based inference, and (iii) estimation of reporting completeness in a sample of nine manuscripts. RESULTS: Ontologies for "food and nutrition" (n = 37), "disease and specific population" (n = 100), "data description" (n = 21), "research description" (n = 35), and "supplementary (meta) data description" (n = 44) were reviewed and listed. ONE consists of 339 classes: 79 new classes to describe data and 24 new classes to describe the content of manuscripts. CONCLUSION: ONE is a resource to automate data integration, searching, and browsing, and can be used to assess reporting completeness in nutritional epidemiology.
Subject(s)
Biological Ontologies/organization & administration , Biomedical Research/standards , Diet , Epidemiologic Methods , Information Dissemination/methods , Nutritional Sciences/standards , Terminology as Topic , Biomedical Research/methods , Data Accuracy , Data Analysis , Humans , Nutritional Sciences/methodsABSTRACT
The RTPrimerDB (http://medgen.ugent.be/rtprimerdb) project provides a freely accessible data retrieval system and an in silico assay evaluation pipeline for real-time quantitative PCR assays. Over the last year the number of user submitted assays has grown to 3500. Data conveyance from Entrez Gene by establishing an assay-to-gene relationship enables the addition of new primer assays for one of the 1.5 million different genes from 2300 species stored in the system. Easy access to the primer and probe data is possible by using multiple search criteria. Assay reports contain gene information, assay details (such as oligonucleotide sequences, detection chemistry and reaction conditions), publication information, users' experimental evaluation feedback and submitter's contact details. Gene expression assays are extended with a scalable assay viewer that provides detailed information on the alignment of primer and probe sequences on the known transcript variants of a gene, along with Single Nucleotide Polymorphisms (SNP) positions and peptide domain information. Furthermore, an mfold module is implemented to predict the secondary structure of the amplicon sequence, as this has been reported to impact the efficiency of the PCR. RTPrimerDB is also extended with an in silico analysis pipeline to streamline the evaluation of custom designed primer and probe sequences prior to ordering and experimental evaluation. In a secured environment, the pipeline performs automated BLAST specificity searches, mfold secondary structure prediction, SNP or plain sequence error identification, and graphical visualization of the aligned primer and probe sequences on the target gene.
Subject(s)
DNA Primers , Databases, Nucleic Acid , Oligonucleotide Probes , Polymerase Chain Reaction/methods , Computer Graphics , DNA Primers/chemistry , Genes , Internet , Oligonucleotide Probes/chemistry , Polymorphism, Single Nucleotide , Terminology as Topic , User-Computer InterfaceABSTRACT
BACKGROUND: DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers. RESULTS: To address this problem we developed methBLAST, a sequence similarity search program, based on the original BLAST algorithm but querying in silico bisulphite modified genome sequences to evaluate oligonucleotide sequence similarities. Apart from the primer specificity analysis tool, we have also developed a public database termed methPrimerDB for the storage and retrieval of validated PCR based methylation assays. The web interface allows free public access to perform methBLAST searches or database queries and to submit user based information. Database records can be searched by gene symbol, nucleotide sequence, analytical method used, Entrez Gene or methPrimerDB identifier, and submitter's name. Each record contains a link to Entrez Gene and PubMed to retrieve additional information on the gene, its genomic context and the article in which the methylation assay was described. To assure and maintain data integrity and accuracy, the database is linked to other reference databases. Currently, the database contains primer records for the most popular PCR-based methylation analysis methods to study human, mouse and rat epigenetic modifications. methPrimerDB and methBLAST are available at http://medgen.ugent.be/methprimerdb and http://medgen.ugent.be/methblast. CONCLUSION: We have developed two integrated and freely available web-tools for PCR based methylation analysis. methBLAST allows in silico assessment of primer specificity in PCR based methylation assays that can be stored in the methPrimerDB database, which provides a search portal for validated methylation assays.
Subject(s)
Computational Biology/methods , DNA Methylation , Polymerase Chain Reaction/methods , Algorithms , Animals , DNA Primers/chemistry , Databases, Genetic , Databases, Protein , Epigenesis, Genetic , Humans , Internet , Mice , Rats , Software , Sulfites/chemistryABSTRACT
The real-time polymerase chain reaction (PCR) methodology has become increasingly popular for nucleic acids detection and/or quantification. As primer/probe design and experimental evaluation is time-consuming, we developed a public database application for the storage and retrieval of validated real-time PCR primer and probe sequence records. The integrity and accuracy of the data are maintained by linking to and querying other reference databases. RTPrimerDB provides free public access through the Web to perform queries and submit user based information. Primer/probe records can be searched for by official gene symbol, nucleotide sequence, type of application, detection chemistry, LocusLink or Single Nucleotide Polymorphism (SNP) identifier, and submitter's name. Each record is directly linked to LocusLink, dbSNP and/or PubMed to retrieve additional information on the gene/SNP for which the primers/probes are designed. Currently, the database contains primer/probe records for human, mouse, rat, fruit fly and zebrafish, and all current detection chemistries such as intercalating dyes (SYBR Green I), hydrolysis probes (Taqman), adjacent hybridizations probes and molecular beacons. Real-time PCR primer/probe records are available at http://www.realtimeprimerdatabase.ht.st.
Subject(s)
DNA Primers/chemistry , Databases, Nucleic Acid , Nucleic Acid Probes/chemistry , Polymerase Chain Reaction , Data Collection , Information Storage and Retrieval , Time FactorsABSTRACT
BACKGROUND: The availability of the human genome sequence as well as the large number of physically accessible oligonucleotides, cDNA, and BAC clones across the entire genome has triggered and accelerated the use of several platforms for analysis of DNA copy number changes, amongst others microarray comparative genomic hybridization (arrayCGH). One of the challenges inherent to this new technology is the management and analysis of large numbers of data points generated in each individual experiment. RESULTS: We have developed arrayCGHbase, a comprehensive analysis platform for arrayCGH experiments consisting of a MIAME (Minimal Information About a Microarray Experiment) supportive database using MySQL underlying a data mining web tool, to store, analyze, interpret, compare, and visualize arrayCGH results in a uniform and user-friendly format. Following its flexible design, arrayCGHbase is compatible with all existing and forthcoming arrayCGH platforms. Data can be exported in a multitude of formats, including BED files to map copy number information on the genome using the Ensembl or UCSC genome browser. CONCLUSION: ArrayCGHbase is a web based and platform independent arrayCGH data analysis tool, that allows users to access the analysis suite through the internet or a local intranet after installation on a private server. ArrayCGHbase is available at http://medgen.ugent.be/arrayCGHbase/.