Cloning, analysis and functional annotation of expressed sequence tags from the Earthworm Eisenia fetida

<p>Abstract</p> <p>Background</p> <p><it>Eisenia fetida</it>, commonly known as red wiggler or compost worm, belongs to the Lumbricidae family of the Annelida phylum. Little is known about its genome sequence although it has been extensively used as a test organism in terrestrial ecotoxicology. In order to understand its gene expression response to environmental contaminants, we cloned 4032 cDNAs or expressed sequence tags (ESTs) from two <it>E. fetida </it>libraries enriched with genes responsive to ten ordnance related compounds using suppressive subtractive hybridization-PCR.</p> <p>Results</p> <p>A total of 3144 good quality ESTs (GenBank dbEST accession number <ext-link ext-link-type="gen" ext-link-id="EH669363">EH669363</ext-link>–<ext-link ext-link-type="gen" ext-link-id="EH672369">EH672369</ext-link> and <ext-link ext-link-type="gen" ext-link-id="EL515444">EL515444</ext-link>–<ext-link ext-link-type="gen" ext-link-id="EL515580">EL515580</ext-link>) were obtained from the raw clone sequences after cleaning. Clustering analysis yielded 2231 unique sequences including 448 contigs (from 1361 ESTs) and 1783 singletons. Comparative genomic analysis showed that 743 or 33% of the unique sequences shared high similarity with existing genes in the GenBank nr database. Provisional function annotation assigned 830 Gene Ontology terms to 517 unique sequences based on their homology with the annotated genomes of four model organisms <it>Drosophila melanogaster</it>, <it>Mus musculus</it>, <it>Saccharomyces cerevisiae</it>, and <it>Caenorhabditis elegans</it>. Seven percent of the unique sequences were further mapped to 99 Kyoto Encyclopedia of Genes and Genomes pathways based on their matching Enzyme Commission numbers. All the information is stored and retrievable at a highly performed, web-based and user-friendly relational database called EST model database or ESTMD version 2.</p> <p>Conclusion</p> <p>The ESTMD containing the sequence and annotation information of 4032 <it>E. fetida </it>ESTs is publicly accessible at <url>http://mcbc.usm.edu/estmd/</url>.</p

Cloning, Analysis and Functional Annotation of Expressed Sequence Tags from the Earthworm \u3ci\u3eEisenia fetida\u3c/i\u3e

Background Eisenia fetida, commonly known as red wiggler or compost worm, belongs to the Lumbricidae family of the Annelida phylum. Little is known about its genome sequence although it has been extensively used as a test organism in terrestrial ecotoxicology. In order to understand its gene expression response to environmental contaminants, we cloned 4032 cDNAs or expressed sequence tags (ESTs) from two E. fetida libraries enriched with genes responsive to ten ordnance related compounds using suppressive subtractive hybridization-PCR. Results A total of 3144 good quality ESTs (GenBank dbEST accession number EH669363–EH672369 and EL515444–EL515580) were obtained from the raw clone sequences after cleaning. Clustering analysis yielded 2231 unique sequences including 448 contigs (from 1361 ESTs) and 1783 singletons. Comparative genomic analysis showed that 743 or 33% of the unique sequences shared high similarity with existing genes in the GenBank nr database. Provisional function annotation assigned 830 Gene Ontology terms to 517 unique sequences based on their homology with the annotated genomes of four model organisms Drosophila melanogaster, Mus musculus, Saccharomyces cerevisiae, and Caenorhabditis elegans. Seven percent of the unique sequences were further mapped to 99 Kyoto Encyclopedia of Genes and Genomes pathways based on their matching Enzyme Commission numbers. All the information is stored and retrievable at a highly performed, web-based and user-friendly relational database called EST model database or ESTMD version 2. Conclusion The ESTMD containing the sequence and annotation information of 4032 E. fetida ESTs is publicly accessible at http://mcbc.usm.edu/estmd

Circular economy and system thinking framework in design; Food system design

The purpose of this thesis is to bring the circular economy framework and system thinking into the process of design thinking. The traditional design process is made up of five steps: 1) Discover a challenge. 2) Interpret the challenge. 3) Create opportunities and ideas. 4) Develop a solution and experiment with it. 5) Evolve the idea. This project is structured upon a methodology generated to apply a circular system framework in design that begins with taking a holistic view of topic. This methodology structured into five phases. The first phase is referring to this approach as moving from Macrocosm to Microcosm; in other words, the mapping of the connections and interconnections of the system. The intersections of these cosmos create what I refer to as the main element, or the “brain” for short (Phase 1). The second phase is to deconstruct this “brain” (Phase 2) and explore the elements within it . The third phase is related with surveying. The fourth phase is defining problems based on the information I found during the past three phases. The last part is the reconstruction of the “brain” using the circular framework (Phase 5)

Batch Blast Extractor: an automated blastx parser application

MotivationBLAST programs are very efficient in finding similarities for sequences. However for large datasets such as ESTs, manual extraction of the information from the batch BLAST output is needed. This can be time consuming, insufficient, and inaccurate. Therefore implementation of a parser application would be extremely useful in extracting information from BLAST outputs. ResultsWe have developed a java application, Batch Blast Extractor, with a user friendly graphical interface to extract information from BLAST output. The application generates a tab delimited text file that can be easily imported into any statistical package such as Excel or SPSS for further analysis. For each BLAST hit, the program obtains and saves the essential features from the BLAST output file that would allow further analysis. The program was written in Java and therefore is OS independent. It works on both Windows and Linux OS with java 1.4 and higher. It is freely available from: http://mcbc.usm.edu/BatchBlastExtractor

Proceedings of the Fourth Annual Conference of the MidSouth Computational Biology and Bioinformatics Society

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A new approach to construct pathway connected networks and its application in dose responsive gene expression profiles of rat liver regulated by 2,4DNT

<p>Abstract</p> <p>Background</p> <p>Military and industrial activities have lead to reported release of 2,4-dinitrotoluene (2,4DNT) into soil, groundwater or surface water. It has been reported that 2,4DNT can induce toxic effects on humans and other organisms. However the mechanism of 2,4DNT induced toxicity is still unclear. Although a series of methods for gene network construction have been developed, few instances of applying such technology to generate pathway connected networks have been reported.</p> <p>Results</p> <p>Microarray analyses were conducted using liver tissue of rats collected 24h after exposure to a single oral gavage with one of five concentrations of 2,4DNT. We observed a strong dose response of differentially expressed genes after 2,4DNT treatment. The most affected pathways included: long term depression, breast cancer regulation by stathmin1, WNT Signaling; and PI3K signaling pathways. In addition, we propose a new approach to construct pathway connected networks regulated by 2,4DNT. We also observed clear dose response pathway networks regulated by 2,4DNT.</p> <p>Conclusions</p> <p>We developed a new method for constructing pathway connected networks. This new method was successfully applied to microarray data from liver tissue of 2,4DNT exposed animals and resulted in the identification of unique dose responsive biomarkers in regards to affected pathways.</p

Enchytraeus albidus Microarray: Enrichment, Design, Annotation and Database (EnchyBASE)

Enchytraeus albidus (Oligochaeta) is an ecologically relevant species used as standard test organisms for risk assessment. Effects of stressors in this species are commonly determined at the population level using reproduction and survival as endpoints. The assessment of transcriptomic responses can be very useful e.g. to understand underlying mechanisms of toxicity with gene expression fingerprinting. In the present paper the following is being addressed: 1) development of suppressive subtractive hybridization (SSH) libraries enriched for differentially expressed genes after metal and pesticide exposures; 2) sequencing and characterization of all generated cDNA inserts; 3) development of a publicly available genomic database on E. albidus. A total of 2100 Expressed Sequence Tags (ESTs) were isolated, sequenced and assembled into 1124 clusters (947 singletons and 177 contigs). From these sequences, 41% matched known proteins in GenBank (BLASTX, e-value≤10-5) and 37% had at least one Gene Ontology (GO) term assigned. In total, 5.5% of the sequences were assigned to a metabolic pathway, based on KEGG. With this new sequencing information, an Agilent custom oligonucleotide microarray was designed, representing a potential tool for transcriptomic studies. EnchyBASE (http://bioinformatics.ua.pt/enchybase/) was developed as a web freely available database containing genomic information on E. albidus and will be further extended in the near future for other enchytraeid species. The database so far includes all ESTs generated for E. albidus from three cDNA libraries. This information can be downloaded and applied in functional genomics and transcription studies

Construction of a cDNA library and preliminary analysis of the expressed sequence tags of the earthworm Eisenia fetida (Savigny, 1826)

Earthworms are useful indicator organisms of soil health and Eisenia fetida have been extensively used as test organisms in ecotoxicological studies. In order to gain insight into the gene expression profiles associated with physiological functions of earthworms, a full‑length enriched cDNA library of the Eisenia fetida genome was successfully constructed using Switching Mechanism at 5\u27End of RNA Template technology. Construction of a cDNA library and analysis of Expressed Sequence Tags (ESTs) are efficient approaches for collecting genomic information and identifying genes important for a given biological process. Furthermore, analysis of the expression abundance of ESTs was performed with the aim of providing genetic and transcriptomic information on the development and regenerative process of earthworms. Phrep and Crossmatch were used to process EST data and a total of 1,140 high‑quality EST sequences were determined by sequencing random cDNA clones from the library. Clustering analysis of sequences revealed a total of 593 unique sequences including 225 contiguous and 368 singleton sequences. Basic Local Alignment Search Tool analysis against the Kyoto Encyclopedia of Genes and Genomes database resulted in 593 significant hits (P‑value \u3c1x10‑8), of which 168 were annotated through Gene Ontology analysis. The STRING database was used to determine relationships among the 168 ESTs, identifying associated genes involved in protein‑protein interactions and gene expression regulation. Based on nucleic acid and protein sequence homology, the mutual relationships between 287 genes could be obtained, which identified a portion of the ESTs as known genes. The present study reports on the construction of a high‑quality cDNA library representative of adult earthworms, on a preliminary analysis of ESTs and on a putative functional analysis of ESTs. The present study is expected to enhance our understanding of the molecular basis underlying the biological development of earthworms