Figure 1. Proposed bioinformatics system architecture .

1. Genomic signatures reveal stressors induced by habitat degradation & climate change in a model reptile species Kurt A Gust1, Mitchell S Wilbanks1, Xianfeng Chen1, Craig A McFarland2, Larry Talent3, Edward J Perkins1 1Environmental Laboratory, US Army, Engineer Research and Development Center, Vicksburg, MS. 2US Army, Public Health Command, Edgewood, MD. 3Oklahoma State University, Stillwater, OK. Full Project Introduction Focus on Genomic Infrastructure Development Table 3.Unigenes homology-based coding potential detection and annotation against the following protein databases: NR.aa (10,606,545 proteins), Refseq (6,392,535 proteins), UniProt-SwissProt (515,203 proteins), Uniref90 (6,544,144 proteins), Uniref100 (9,865,668 proteins). • Web Accessible Knowledgebase: We have implemented a mature bioinformatics and computational biology system which includes: • (1) a Relational Database Management Systems (RDBMS) - Oracle (Oracle, Redwood Shores, CA) for quick data retrieval and integration • (2) public and private data and results access via network shared file servers (http://jeff.ifxworks.com/EGGT/Quail_Lizard.html) • (3) data and results visualization via a public accessible web server (http://www.ifxworks.com/EnvironmentalSystemsBiology.html) Materials and Methods: • WFL normalized-cDNA Library Construction • RNA extracted from brain, liver, gut, heart, bone marrow and gonad tissues. • Tissues Collected from 5 male and 5 female, unexposed WFL. • All RNA quality assured using gel-electrophoresis. • SMART™ PCR cDNA Synthesis used to reverse transcribe full length cDNAs • Trimmer, cDNA Normalization Kit used to normalize cDNA library. Sequencing, Annotation & Microarray Development • (4) High performance and throughput computational analysis pipelines for quick data loading, retrieval, analysis, processing, integration, and validation (Figure 1 and 2). ANNOTATION Roche - GS-FLX 929K Sequences • Diamond Supercomputer at ERDC Agilent G3 Custom 60K oligonucleotide microarray Results and Discussion: Table 1. Results of GS-FLX Pyrosequencing of normalized cDNA Library for Western fence lizard (WFL). • Problem Identification: • Multiple-stressor effects of common and emerging environmental stressors are unknown for native populations including the reptile model Western Fence Lizard (WFL). • As habitat loss and global warming advance, resulting stressors may exacerbate chemical stressors (MECs) on Army Ranges. • Tools are needed to forensically identify predominant environmental stressors to optimize field management. • Purpose: • Assess and characterize the effects of chemical stress (TNT) in conjunction with environmental stressors induced by habitat loss and climate change in a model reptile species. • Null Hypotheses:A.Multiple ecosystem-level stressors characteristic of habitat degradation and climate change have no interactive effects on lizard health and fitness. B.Environmental stressors are uniquely identifiable via genomic signatures and these signatures can be used to identify predominant stressors in multiple-stressor scenarios. • Sequencing: 1 GS-FLX pyrosequencing run yielded 329 megabases in 929K reads with 354bp average read length (Table 1). • Clustering: A genome-scale transcriptome for WFL was used for EST-based clustering and assembly via The Gene Indices Clustering Tools (TGICL), which uses megablast for homology-based clustering and CAP3. • Annotation: In all, 53,897 contigs and 5,065 singlets totaling 58,962 unigenes were identified (Table 2). Approx 44 % of unigenes were annotated for protein-coding potential via homology-based annotation against NCBI NR.aa, Refseq, and EBI UniProt-SwissProt, Uniref90, and Uniref100 protein sequence reference knowledgebases (Table 3). • Microarray Design – Transition WFL transcriptome to Agilent G3 Custom 60K oligonucleotidemicroarray. Figure 1.Proposed bioinformatics system architecture. • Conclusions & Future Efforts: • We are the first to develop gene expression tools and a publicly accessible, extensive genomics infrastructure for a laboratory-amenable reptile-model species, the Western Fence Lizard. • These assets will enable the in progress assessment of emerging stressors that are directly influenced by the non-point multi-stakeholder impacts, habitat loss and climate change (See Platform Pres. # 386). Table 2. Summary of sequence clustering and assembly for Western Fence Lizard (WFL). Figure 2.Web dissemination of datasets including web-based tools for transcriptomes and unigeneanalysis. http://jeff.ifxworks.com/EGGT/Quail_Lizard.html