North East Cyberinfrastructure Consortium

1. Comparative Analysis of Three Cartilaginous Fish Transcriptomes To Advance Studies of Human Gene Function North East Bioinformatics Consortium of The North East Cyberinfrastructure Consortium Abstract Background Transcriptome Sequencing, Assembly and Annotation Background and Objective Comparative functional genomics research seeks to obtain a deeper understanding of human genes by studying the function of related genes in multiple organisms. Although mammalian model organisms are powerful, more diverse vertebrate models provide significant advantages to study conserved genes and pathways. Studies in cartilaginous fishes are of particular importance as they are the most basal extant jawed vertebrates with adaptive immune systems and fundamental vertebrate characteristics that have been used to advance studies of human physiology, stem cell biology, immunology, toxicology and development. We sequenced, assembled and annotated the skate (Leucoraja erinacea) transcriptome and compared it two other cartilaginous fishes to study the conservation of human genes over ~450 million years of evolution. Methods We sequenced, assembled and annotated the skate transcriptome using Illumina sequencing of three individual embryos with and compared it to Scyliorhinus canicula, Callorhincus milii and human transcriptomes. Results 99.2% of core eukaryotic genes were found in the skate and 11,102 human genes have predicted orthologs. We analyzed genes involved in hematopoietic stem cell production and the ornithine-urea cycle because of unique biological characteristics of these organisms. Discussion and Conclusions The skate transcriptome is a valuable resource to study conserved genes, pathways and biological processes, such as hematopoiesis and osmoregulation. Transcriptome Sequencing (RNA-Seq) and Assembly Cartilaginous Fishes Most Basal Extant Jawed Vertebrates Trinity Assemblies Randomly Fragment cDNA Made From RNA Cartilaginous fishes (chondrichthyes) diverged approximately 450 million years ago, closely following the initial appearance of jaws and paired appendages in the vertebrate fossil record. They exhibit fundamental vertebrate characteristics including adaptive immune systems. Sequence Fragments Skate Is An Informative Comparative Model Assembly ...ATCCGTAAATGGGCTGATACTACTAATGC TGGGCTGATACTACTAATGCCAAACTGTACTAGTCCTG... ...ATCCGTAAATGGGCTGATACTACTAATGCCAAACTGTACTAGTCCTG... • - Renal physiology • Stem cell biology • Immunology • Development • - Toxicology Elasmobranchs Contiguous Sequence (Contig) Triannotate Annotation Pipeline Amemiya CT et al. Nature (2013) 496(7445):311-6 • North East Cyberinfrastructure Consortium • The NECC successfully built regional cyberinfrastructure and conducted coordinated cyber-enabled bioinformatics research and training through its ongoing skate genome project. • INBRE Programs in: • - Maine • Delaware • Vermont • New Hampshire • Rhode Island • Goals: • 1. Characterize the skate transcriptome of late-stage embryos • 2. Compare it to transcriptomes of late-stage embryos from two other cartiliginaous fishes: • Catshark (S. canicula) • Elephant shark (C. milii) • 3. Examine conservation of human genes involved with: • Hematopoiesis • Osmoregulation Illumina RNA-Seq Data Leucoraja erinacea Scyliorhinus canicula Callorhinchus milii Leucoraja erinacea Scyliorhinus canicula Chimaera/Holocephalon http://skatebase.org http://www.necyberconsortium.org Callorhinchus milii Comparisons of Protein Coding Genes Clustering Hematopoiesis Osmoregulation Protein Domains and Pathways Clustering of Predicted with Known Proteins Predicted Orthologs Hematopoiesis in the Skate Osmoregulation Hematopoiesis Genes Under Evaluation in Transcriptomes Putative orthologous gene groupings were identified using OrthoDB. Skate, catshark and elephant shark transcripts were mapped to OrthoDB ortholog groups using shared UniProt protein sequence identifiers reported by Triannotate. InterProScan5 was used to identify protein domains among the predicted open reading frames. The UniProt Reference 90 (UniRef90) resource provides a clustered set of known protein sequences from the UniProt Knowledgebase that align with one another with greater than 90% identity over at least 80% of the length using CD-HIT. Skate, catshark and elephant shark transcripts were clustered with UniRef90 using CD-HIT2 using an 80% overlap threshold. Transcripts were combined in seven different combinarions as shown below (e.g., skate only (XXX_Ler_XXX), catshark and elephant shark only (Cmil_XXX_Sca), etc.) The skate is a valuable model system to study multipotent hematopoietic stem and progenitor cells (HPSC) that produce all blood cell lineages. The complex microenvironment in which HPSC reside is difficult to study in mammals as they reside in the bone portion of the marrow (i.e., the endosteal niche). In contrast, HPSC are easily accessible in elasmobranch fishes, including the skate, as they reside in the epigonal and Leydig organs. The epigonal organ is in direct contact with the gonads and offers a unique system to study the HPSC microenvironment as the organ undergoes significant angiogenesis and HSPC mobilization during reproductive. Elasmobranchs, including the skate, have been extensively studied as models for renal physiology because they retain urea in their circulatory systems to maintain osmotic homeostasis in sea water. Ornithine-Urea Cycle (OUC) Genes We are currently examining the expression of OUC enzymes in the transcriptomes. Summary of identified domains and their representation in metabolic and signaling pathways. Comparison of Skate and Human Genes 11,102 of 19,536 Ensembl human protein-coding genes belong to orthologous groups mapped to the skate. Expression of CXCR4 and CXCL12 confirmed by PCR in skate epigonal organ 6,701 of 8,699 Ensembl human protein-coding genes with lamprey orthologs belong to orthologous groups mapped to the skate. Confirmed by Taylor Hersh in the Laboratory of Prof. Bram Lutton (MDIBL) Trumpp et al, Nat Review Immunol. (2010) 10(3):201-9 Image credit: http://ureacycle.cnmcresearch.org/otc/ Future Directions Funding Continue to collaboratively: 1. Work on genome and transcriptome sequencing, assembly and annotation. 2. Develop and deliver bioinformatics training materials. 3. Share regional computational and data storage infrastructure. Skate genome sequencing was funded by: NIH NCRR ARRA Supplements to 5 P20 RR016463-12 (MDIBL), 5 P20 RR016472-12 (UD), 5 P20 RR16462 (UVM). The North East Cyberinfrasturcture Consortium is funded by: - NIH National Center for Research Resources grants: 5 P20 RR016463-12 (MDIBL), 5 P20 RR016472-12 (UD), 5 P20 RR16462 (UVM), 5 P20 RR016457-11 (URI), 5 P20 RR030360-03 (UNH) - NIH National Institute of General Medical Sciences grants: 8 P20 GM103423-12 (MDIBL), 8 P20 GM103446-12 (UD), 8 P20 GM103449 (UVM), 8 P20 GM103430-11 (URI), 8 P20 GM103506-03 (Dartmouth) - National Science Foundation EPSCoR grants: EPS-0904155 (UM), EPS-081425 (UD), EPS-1101317 (UVM), EPS-1004057 (URI), EPS-1101245 (UNH).