Abstract
The skin of fish is the first line of defense against pathogens and parasites. The skin transcriptome of the Atlantic salmon is poorly characterized, and currently only 2,089 expressed sequence tags (ESTs) out of a total of half a million sequences are generated from skin-derived cDNA libraries. The primary aim of this study was to enhance the transcriptomic knowledge of salmon skin by using next-generation sequencing (NGS) technology, namely the Roche-454 platform. An equimolar mixture of high-quality RNA from skin and epidermal samples of salmon reared in either freshwater or seawater was used for 454-sequencing. This technique yielded over 600,000 reads, which were assembled into 34,696 isotigs using Newbler. Of these isotigs, 12 % had not been sequenced in Atlantic salmon, hence representing previously unreported salmon mRNAs that can potentially be skin-specific. Many full-length genes have been acquired, representing numerous biological processes. Mucin proteins are the main structural component of mucus and we examined in greater detail the sequences we obtained for these genes. Several isotigs exhibited homology to mammalian mucins (MUC2, MUC5AC and MUC5B). Mucin mRNAs are generally >10 kbp and contain large repetitive units, which pose a challenge towards full-length sequence discovery. To date, we have not unearthed any full-length salmon mucin genes with this dataset, but have both N- and C-terminal regions of a mucin type 5. This highlights the fact that, while NGS is indeed a formidable tool for sequence data mining of non-model species, it must be complemented with additional experimental and bioinformatic work to characterize some mRNA sequences with complex features.
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References
Adzhubei AA, Vlasova AV, Hagen-Larsen H, Ruden TA, Laerdahl JK, Høyheim B (2007) Annotated expressed sequence tags (ESTs) from pre-smolt Atlantic salmon (Salmo salar) in a searchable data resource. BMC Genomics 8:209–224
Alexander JB, Ingram GA (1992) Noncellular nonspecific defence mechanisms of fish. Annu Rev Fish Dis 2(C):249–279
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410
Alvarez-Pellitero P (2008) Fish immunity and parasite infections: from innate immunity to immunoprophylactic prospects. Vet Immunol Immunopathol 126(3–4):171–198
Barrell D, Dimmer E, Huntley RP, Binns D, O'Donovan C, Apweiler R (2009) The GOA database in 2009 — an integrated gene ontology annotation resource. Nucleic Acids Res 37(suppl 1):D396–D403
Bettencourt R, Pinheiro M, Egas C et al (2010) High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus. BMC Genomics 11:559–576
Bhat GG, Shetty KN, Nagre NN et al (2010) Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity. Glycoconj J 27(3):309–320
Bostock J, McAndrew B, Richards R et al (2010) Aquaculture: Global status and trends. Philos Trans R Soc B Biol Sci 365(1554):2897–2912
Cárdenas L, Sánchez R, Gomez D, Fuenzalida G, Gallardo-Escárate C, Tanguy A (in press) Transcriptome analysis in Concholepas concholepas (Gastropoda, Muricidae): mining and characterization of new genomic and molecular markers. Mar Genomics
Clark MS, Thorne MAS, Vieira FA, Cardoso JCR, Power DM, Peck LS (2010) Insights into shell deposition in the Antarctic bivalve Laternula elliptica: gene discovery in the mantle transcriptome using 454 pyrosequencing. BMC Genomics 11(1):362–376
Davidson WS, Koop BF, Jones SJM et al (2010) Sequencing the genome of the Atlantic salmon (Salmo salar). Genome Biol 11(9):403–410
Droege M, Hill B (2008) The genome sequencer FLX™ system — longer reads, more applications, straight forward bioinformatics and more complete data sets. J Biotechnol 136(1–2):3–10
Easy RH, Ross NW (2009) Changes in Atlantic salmon (Salmo salar) epidermal mucus protein composition profiles following infection with sea lice (Lepeophtheirus salmonis). Comp Biochem Physiol D Genomics Prot 4(3):159–167
Ekblom R, Galindo J (2011) Applications of next generation sequencing in molecular ecology of non-model organisms. Heredity 107(1):1–15
Elliott DG (2000) Integumentary system. In: Ostrander GK (ed) The laboratory fish, 1st edn. Academic Press, CA, pp 271–306
Finstad AG, Einum S, Forseth T, Ugedal O (2007) Shelter availability affects behaviour, size-dependent and mean growth of juvenile Atlantic salmon. Freshw Biol 52(9):1710–1718
Goetz F, Rosauer D, Sitar S et al (2010) A genetic basis for the phenotypic differentiation between siscowet and lean lake trout (Salvelinus namaycush). Mol Ecol 19(suppl 1):176–196
Gum JR, Byrd JC, Hicks JW, Toribara NW, Lamport DTA, Kim YS (1989) Molecular cloning of human intestinal mucin cDNAs. sequence analysis and evidence for genetic polymorphism. J Biol Chem 264(11):6480–6487
Harris JE, Hunt S (1975) The fine structure of the epidermis of two species of salmonid fish, the Atlantic salmon (Salmo salar L.) and the brown trout (Salmo trutta L.): II. mucous cells. Cell Tissue Res 163(4):535–543
Horreo JL, Machado-Schiaffino G, Ayllon F et al (2011) Impact of climate change and human-mediated introgression on southern European Atlantic salmon populations. Global Change Biol 17(5):1778–1787
Hou R, Bao Z, Wang S et al (2011) Transcriptome sequencing and de novo analysis for Yesso scallop (Patinopecten yessoensis) using 454 GS FLX. PLoS One 6(6):e21560
Hu Z, Bao J, Reecy JM (2008) CateGOrizer: a web-based program to batch analyze gene ontology classification categories. Online J Bioinf 9(2):108–112
Huang Z, Ma A, Wang X (2011) The immune response of turbot, Scophthalmus maximus (L.), skin to high water temperature. J Fish Dis 34(8):619–627
Jeukens J, Renaut S, St-Cyr J, Nolte AW, Bernatchez L (2010) The transcriptomics of sympatric dwarf and normal lake whitefish (Coregonus clupeaformis spp., Salmonidae) divergence as revealed by next-generation sequencing. Mol Ecol 19(24):5389–5403
Kircher M, Kelso J (2010) High-throughput DNA sequencing — concepts and limitations. Bioessays 32(6):524–536
Koop BF, Von Schalburg KR, Leong J et al (2008) A salmonid EST genomic study: genes, duplications, phylogeny and microarrays. BMC Genomics 9:545–561
Kumar S, Blaxter ML (2010) Comparing de novo assemblers for 454 transcriptome data. BMC Genomics 11:571–583
Lang T, Alexandersson M, Hansson GC, Samuelsson T (2004) Bioinformatic identification of polymerizing and transmembrane mucins in the puffer fish Fugu rubripes. Glycobiology 14(6):521–527
Leong JS, Jantzen SG, von Schalburg KR et al (2010) Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome. BMC Genomics 11:279–296
Løvoll M, Wiik-Nielsen J, Grove S et al (2010) A novel totivirus and piscine reovirus (PRV) in Atlantic salmon (Salmo salar) with cardiomyopathy syndrome (CMS). Virol J 7:309–316
Martin JA, Wang Z (2011) Next-generation transcriptome assembly. Nat Rev Genet 12(10):671–682
Martin SAM, Taggart JB, Seear P et al (2007) Interferon type I and type II responses in an Atlantic salmon (Salmo salar) SHK-1 cell line by the salmon TRAITS/SGP microarray. Physiol Genomics 32(1):33–44
Miller JR, Koren S, Sutton G (2010) Assembly algorithms for next-generation sequencing data. Genomics 95(6):315–327
Moniaux N, Escande F, Porchet N, Aubert JP, Batra SK (2001) Structural organization and classification of the human mucin genes. Front Biosci 6:D1192–D1206
Myers EW, Sutton GG, Delcher AL et al (2000) A whole-genome assembly of Drosophila. Science 287(5461):2196–2204
Palaksha KJ, Shin G, Kim Y, Jung T (2008) Evaluation of non-specific immune components from the skin mucus of olive flounder (Paralichthys olivaceus). Fish Shellfish Immunol 24(4):479–488
Phillipson M, Johansson MEV, Henriksnäs J et al (2008) The gastric mucus layers: constituents and regulation of accumulation. Am J Physiol Gastrointest Liver Physiol 29(4):G806–G812
Rise ML, von Schalburg KR, Brown GD et al (2004) Development and application of a salmonid EST database and cDNA microarray: data mining and interspecific hybridization characteristics. Genome Res 14(3):478–490
Rose MC, Voynow JA (2006) Respiratory tract mucin genes and mucin glycoproteins in health and disease. Physiol Rev 86(1):245–278
Salem M, Rexroad CE III, Wang J, Thorgaard GH, Yao J (2010) Characterization of the rainbow trout transcriptome using Sanger and 454-pyrosequencing approaches. BMC Genomics 11:564–574
Tadiso TM, Krasnov A, Skugor S, Afanasyev S, Hordvik I, Nilsen F (2011) Gene expression analyses of immune responses in Atlantic salmon during early stages of infection by salmon louse (Lepeophtheirus salmonis) revealed bi-phasic responses coinciding with the copepod-chalimus transition. BMC Genomics 12:141–158
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739
Thornton DJ, Rousseau K, McGuckin MA (2008) Structure and function of the polymeric mucins in airways mucus. Annu Rev Physiol 70:459–486
Thorsen J, Zhu B, Frengen E et al (2005) A highly redundant BAC library of Atlantic salmon (Salmo salar): an important tool for salmon projects. BMC Genomics 6:50–58
Tsutsui S, Tasumi S, Suetake H, Suzuki Y (2003) Lectins homologous to those of monocotyledonous plants in the skin mucus and intestine of pufferfish, Fugu rubripes. J Biol Chem 278(23):20882–20889
Vera JC, Wheat CW, Fescemyer HW et al (2008) Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing. Mol Ecol 17(7):1636–1647
Verma M, Davidson EA (1993) Molecular cloning and sequencing of a canine tracheobronchial mucin cDNA containing a cysteine-rich domain. Proc Natl Acad Sci USA 90(15):7144–7148
Welinder KG, Tams JW (1995) Effects of glycosylation on protein folding, stability and solubility. studies of chemically modified or engineered plant and fungal peroxidases. Carbohydr Bioeng 10:205–210
Xiang L, He D, Dong W, Zhang Y, Shao J (2010) Deep sequencing based transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus reveals insight into the immune-relevant genes in marine fish. BMC Genomics 11:472–493
Zhou X, Ren L, Meng Q, Li Y, Yu Y, Yu J (2010) The next-generation sequencing technology and application. Protein Cell 1(6):520–536
Acknowledgements
The research was supported by a studentship to G. Micallef funded by BioMar Ltd. The authors would like to thank Dr. William Roy and Dr. Andrew Tildesley for their assistance with fish handling at the MERL in Machrihanish.
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A.S. Bowman and S.A.M. Martin contributed equally to the work.
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Micallef, G., Bickerdike, R., Reiff, C. et al. Exploring the Transcriptome of Atlantic Salmon (Salmo salar) Skin, a Major Defense Organ. Mar Biotechnol 14, 559–569 (2012). https://doi.org/10.1007/s10126-012-9447-2
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DOI: https://doi.org/10.1007/s10126-012-9447-2