Toxicogenomics analysis of non-model transcriptomes using next-generation sequencing and microarray.

摘要

With the advent of next generation technologies like Roche/454 Life Sciences that require low cost and less time for sequencing will help in providing a workable draft of non-model species genomes. Availability of high throughput microarray technologies for gene expression profiling provides low-cost tools for investigation of highly-integrated responses to various stimuli. These advancements along with bioinformatics processing have led to an increasing number of non-model species having well-annotated transcriptomes. The project focuses on the life cycle of development, functional annotation, and utilization of genomic tools for the avian wildlife species to determine the molecular impacts of exposure to munitions constituents (MCs).;Massively parallel pyrosequencing is created from the normalized multi-tissue library of Northern bobwhite (Colinus virginianus) and Japanese quail (Coturnix coturnix) cDNAs. The assembly of next generation sequencing for transcriptomes of these organisms is challenging. High number of ESTs and longer read length require high computational memory and management between the sensitivity and accuracy to assemble correctly. The researcher developed a new pipeline “Contigs Assembly Pipeline using Reference Genome” (CAPRG) to assemble long reads for non-model organisms that have available reference genome. The results were benchmarked by employing parameter space for different available methods that utilize de novo strategies like overlap-layout-consensus (OLC) and graphs for long reads. It was observed that CAPRG performance was better or near equivalent in the two transcriptomic datasets based on different benchmarks but also completes the assembly in a fraction of the time as compared to assemblers that yield competitive results.;The researcher performed statistical analysis to generate differentially expressed genes and utilized metabolic maps, biological networks, pathway analysis and GO enrichment to the differentially-expressed genes in the livers of birds exposed for 60 days (d) to 10 and 60 mg/kg/d 2,6-DNT. These revealed insights into the metabolic perturbations underlying several observed toxicological phenotypes. The impacts were validated by RT-qPCR including: a shift in energy metabolism toward protein catabolism via inhibition of control points for glucose and lipid metabolic pathways, PCK1 and PPARGC1, respectively.;To greatly expand the information-base for Northern bobwhite that has little supporting information in Genbank, the researcher initiated and developed the web-based knowledgebase (www.quailgenomics.info). The Quail genomics share and develop functional genomic data for Northern bobwhite to allow researchers to perform analysis and curate genomic information for this non-model species.