Marine Genomics 23 (2015) 37–40
Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Genomics/Technical resources
Transcriptome analysis of gill tissue of Atlantic cod Gadus morhua L. from the Baltic Sea Magdalena Małachowicz, Agnieszka Kijewska, Roman Wenne ⁎ Institute of Oceanology PAS, 81-712 Sopot, Poland
a r t i c l e
i n f o
Article history: Received 23 March 2015 Received in revised form 15 April 2015 Accepted 15 April 2015 Available online 24 April 2015 Keywords: Atlantic cod Baltic 454 pyrosequencing Transcriptome
a b s t r a c t The Atlantic cod (Gadus morhua L.) is one of the most ecologically and economically important marine fish species in the North Atlantic Ocean. Using Roche GS-FLX 454 pyrosequencing technique 962,516 reads, representing 379 Mbp of the Baltic cod transcriptome, were obtained. Data was assembled into 14,029 contigs of which 100% displayed homology to the Atlantic cod transcriptome. Despite a high similarity between transcripts, evidence for significant differences between Baltic and Atlantic cod was found. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
2. Data description
The Atlantic cod (Gadus morhua L.) is a key species in the North Atlantic Ocean, with significant ecological and commercial value. It lives at low-salinity (7–12‰) in the Baltic Sea, but requires salinity over 14‰ for successful spawning (Nissling and Westin, 1997). Areas suitable for reproduction of cod are accessible in a few zones only in the Baltic Sea (Bornholm, Belt Sea) thanks to rare intrusions of high salinity and well oxygenated water from the North Sea. The Baltic cod produces eggs with thinner chorion compared to fish from outside the Baltic as a result of adaptation to low salinity (Nissling et al., 1994). The Baltic and Atlantic populations differ genetically (Nielsen et al., 2003; O'Leary et al., 2007; Poćwierz-Kotus et al., 2015), but current understanding of Baltic cod genetics is still limited. Several Atlantic cod transcriptomes obtained from eggs and embryos have been characterized with micro-arrays (Rise et al., 2014; Škugor et al., 2014). Furthermore, Roche GS-FLX 454 pyrosequencing based transcriptomes from different tissues (brain, gonad, head kidney, hindgut, liver, spleen) and eggs have been published (Star et al., 2011; Lanes et al., 2013). However, no transcriptome data is currently available for Atlantic cod from the Baltic Sea. The aim of this study was to assemble, annotate and analyze Baltic cod transcriptome from gills and compare it to the Atlantic cod available data from the Cod Genome Project.
2.1. RNA preparation and sequencing Atlantic cod were sampled in November 2011 from the Schleifjord, Kiel Bight (KIEL) and Gdańsk Bay (GDA). Live fish were transported to the Marine Station of the University of Gdańsk in Hel and settled in tanks (3500 l). They were kept at 10 °C in water which simulated the natural salinities of the geographic source region of the cod (KIEL — 18‰ and GDA — 8‰). The fish were maintained at natural photoperiod and acclimated to laboratory conditions for a minimum of 14 days until they start feeding and displayed typical behaviour. All experiments complied with EC Directive 2010/63/EU for animal experiments and with the permission no. 60/2012 of the Local Ethics Committee on Animal Experimentation No. 3. Samples of gills were collected and stored in RNAlater, following the manufacturer's instruction (Quiagen, Hilden, Germany). Total RNA from 36 individuals was extracted using the ISOLATE II RNA Mini Kit (Bioline, London, UK) and stored at − 20 °C. Concentration of extracted RNA was determined at 260 nm on microplate using the Epoch Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, USA). Samples were processed using a 454 pyrosequencing by CD Genomics (USA). 2.2. Transcriptome assembly
⁎ Corresponding author. Tel.: +48 58 731 17 63; fax: +48 58 551 21 30. E-mail address: [email protected] (R. Wenne).
After trimming adapters and low quality bases, a total of 962,516 reads with mean length 300–400 bp (Fig. 1) were used for de novo
http://dx.doi.org/10.1016/j.margen.2015.04.005 1874-7787/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
38
M. Małachowicz et al. / Marine Genomics 23 (2015) 37–40
2.3. Functional annotation
Fig. 1. Frequency distribution of raw reads lengths on a logarithmic scale.
transcriptome assembly using the CLC Genomics Workbench ver. 7.5.1, CLC Bio, Aarhus, Denmark (see Supplementary methods for details).
B
Of the 14,029 contigs, 100% homologous transcripts (13,585 genes) were identified using BLASTn with an E-value threshold of 10 − 10. All contigs were divided into biotypes (Fig. 2A). Gene ontology (GO) terms were assigned to each contig using the Blast2GO tool (www.blast2go.com, Götz et al., 2008). Among 13,585 genes 75.39% had an ontology definition and were classified into three main GO categories (biological process — BP, molecular function — MF and cellular component — CC) and 45 subcategories (Fig. 2B). Among these genes, 48.34% were assigned to at least one GO term in the molecular function category, 33.24% in biological processes, and 18.42% in cellular component (see Supplementary methods for details). In addition, we compared Baltic cod contigs with the Atlantic Ocean cod reference transcriptome (http://www.ensembl.org/Gadus_morhua) using BLASTn. The identified Baltic cod transcripts covered 58% of the Atlantic cod transcriptome (Fig. 3A). While 13,827 (98.56%) Baltic transcripts aligned perfectly and unambiguously, 202 (1.44%; 170 genes) demonstrated significant distinctiveness from Atlantic cod (Fig. 3B). Among those 170 genes 100% were protein coding sequences; 68.82%
A
Fig. 2. Summary of Baltic cod transcriptome assembly. (A) Among 14,029 contigs 13,704 were coding proteins, 240 were annotated as pseudogenes, and 85 as small noncoding RNAs (logarithmic scale). (B) A total of 10,242 genes were assigned to at least one GO term and were grouped into three main categories and 45 subcategories.
M. Małachowicz et al. / Marine Genomics 23 (2015) 37–40
A
39
B
C
Fig. 3. Comparison of Baltic and Atlantic cod using BLASTn and Blast2Go. (A) Baltic cod transcripts covered 58% of Atlantic cod transcriptome. (B) 1.44% (202 transcripts, 170 genes) demonstrated differences between Baltic and Atlantic cod. (C) Within these genes, 56.63% belonged to molecular function, 28.06% to biological process and 15.61% cellular component.
of them had an ontology definition and were classified into three main GO categories (BP, MF, CC) and 29 subcategories (Fig. 3C). The molecular function classes was the most highly represented (56.63%), followed by biological process (28.06%) and cellular component (15.31%) (see Supplementary methods for details). While exhibiting similarities on a broad scale, evidence of significant differences between transcriptomes from the two samples of Atlantic cod from the Baltic Sea and Atlantic cod reference was found (Fig. 3B). These differences could have been caused by geographic origin from different native waters, which confirms the need for a reference transcriptome for Baltic cod.
Acknowledgements
2.4. Nucleotide sequence accession numbers
References
The Baltic cod raw data from this study were deposited in the NCBI Sequence Read Archive under the accession number [SRP052904]. Assembled contigs and annotation information are available from the authors upon request.
This study was partially funded by project: 2011/01/M/NZ9/07207 of the National Science Centre in Poland to RW and statutory topic IV.1 in the IO PAS. We would like to thank dr. rer. nat. Ch. Petereit (GEOMAR) for fish collection from Kiel Bight, Germany. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.margen.2015.04.005.
Götz, S., Garcia-Gómez, J.M., Terol, J., Williams, T.D., Nagaraj, S.H., Nueda, M.J., Robles, M., Talón, M., Dopazo, J., Conesa, A., 2008. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res. 36, 3420–3435. http://dx. doi.org/10.1093/nar/gkn176. Lanes, C.F.C., Bizuayehu, T.T., de Oliveira Fernandes, J.M., Kiron, V., Babiak, I., 2013. Transcriptome of Atlantic Cod (Gadus morhua L.) Early embryos from farmed and wild
40
M. Małachowicz et al. / Marine Genomics 23 (2015) 37–40
broodstock. Mar. Biotechnol. 15, 677–694. http://dx.doi.org/10.1007/s10126-0139527-y. Nielsen, E.E., Hansen, M.M., Ruzzante, D.E., Meldrup, D., Grønkjær, P., 2003. Evidence of a hybrid-zone in Atlantic cod (Gadus morhua) in the Baltic and the Danish Belt Sea revealed by individual admixture analysis. Mol. Ecol. 12, 1497–1508. http://dx.doi.org/ 10.1046/j.1365-294X.2003.01819.x. Nissling, A., Westin, L., 1997. Salinity requirements for successful spawning of Baltic and Belt Sea cod and the potential for cod stock interactions in the Baltic Sea. Mar. Ecol. Prog. Ser. 152, 261–271. Nissling, A., Kryvi, H., Vallin, L., 1994. Variation in egg buoyancy of Baltic cod (Gadus morhua) and its implications for egg survival in prevailing conditions in the Baltic Sea. Mar. Ecol. Prog. Ser. 110, 67–74. O'Leary, D.B., Coughlan, J., Dillane, E., McCarthy, T.V., Cross, T.F., 2007. Microsatellite variation in cod Gadus morhua throughout its geographic range. J. Fish Biol. 70, 310–335. http://dx.doi.org/10.1111/j.1095-8649.2007.01451.x.
Poćwierz-Kotus, A., Kijewska, A., Petereit, C., Bernaś, R., Więcaszek, B., Arnyasi, M., Lien, S., Kent, M.P., Wenne, R., 2015. Genetic differentiation of brackish water populations of cod Gadus morhua in the southern Baltic, inferred from genotyping using SNP-arrays. Mar. Genomics 19, 17–22. http://dx.doi.org/10.1016/j.margen.2014.05.010. Rise, M.L., Nash, G.W., Hall, J.R., Booman, M., Hori, T.S., Trippel, E.A., Gamperl, A.K., 2014. Variation in embryonic mortality and maternal transcript expression among Atlantic cod (Gadus morhua) broodstock: a functional genomics study. Mar. Genomics 18 (Pt A), 3–20. http://dx.doi.org/10.1016/j.margen.2014.05.004. Škugor, A., Krasnov, A., Andersen, Ø., 2014. Genome-wide microarray analysis of Atlantic cod (Gadus morhua) oocyte and embryo. BMC Genomics 15, 594. http://dx.doi.org/ 10.1186/1471-2164-15-594. Star, B., Nederbragt, A.J., Jentoft, S., Grimholt, U., Malmstrom, M., Gregers, T.F., Rounge, T.B., Paulsen, J., Solbakken, M.H., Sharma, A., et al., 2011. The genome sequence of Atlantic cod reveals a unique immune system. Nature 477 (7363), 207–210. http:// dx.doi.org/10.1038/nature10342.
Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Genomics/Technical resources
Transcriptome analysis of gill tissue of Atlantic cod Gadus morhua L. from the Baltic Sea Magdalena Małachowicz, Agnieszka Kijewska, Roman Wenne ⁎ Institute of Oceanology PAS, 81-712 Sopot, Poland
a r t i c l e
i n f o
Article history: Received 23 March 2015 Received in revised form 15 April 2015 Accepted 15 April 2015 Available online 24 April 2015 Keywords: Atlantic cod Baltic 454 pyrosequencing Transcriptome
a b s t r a c t The Atlantic cod (Gadus morhua L.) is one of the most ecologically and economically important marine fish species in the North Atlantic Ocean. Using Roche GS-FLX 454 pyrosequencing technique 962,516 reads, representing 379 Mbp of the Baltic cod transcriptome, were obtained. Data was assembled into 14,029 contigs of which 100% displayed homology to the Atlantic cod transcriptome. Despite a high similarity between transcripts, evidence for significant differences between Baltic and Atlantic cod was found. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
2. Data description
The Atlantic cod (Gadus morhua L.) is a key species in the North Atlantic Ocean, with significant ecological and commercial value. It lives at low-salinity (7–12‰) in the Baltic Sea, but requires salinity over 14‰ for successful spawning (Nissling and Westin, 1997). Areas suitable for reproduction of cod are accessible in a few zones only in the Baltic Sea (Bornholm, Belt Sea) thanks to rare intrusions of high salinity and well oxygenated water from the North Sea. The Baltic cod produces eggs with thinner chorion compared to fish from outside the Baltic as a result of adaptation to low salinity (Nissling et al., 1994). The Baltic and Atlantic populations differ genetically (Nielsen et al., 2003; O'Leary et al., 2007; Poćwierz-Kotus et al., 2015), but current understanding of Baltic cod genetics is still limited. Several Atlantic cod transcriptomes obtained from eggs and embryos have been characterized with micro-arrays (Rise et al., 2014; Škugor et al., 2014). Furthermore, Roche GS-FLX 454 pyrosequencing based transcriptomes from different tissues (brain, gonad, head kidney, hindgut, liver, spleen) and eggs have been published (Star et al., 2011; Lanes et al., 2013). However, no transcriptome data is currently available for Atlantic cod from the Baltic Sea. The aim of this study was to assemble, annotate and analyze Baltic cod transcriptome from gills and compare it to the Atlantic cod available data from the Cod Genome Project.
2.1. RNA preparation and sequencing Atlantic cod were sampled in November 2011 from the Schleifjord, Kiel Bight (KIEL) and Gdańsk Bay (GDA). Live fish were transported to the Marine Station of the University of Gdańsk in Hel and settled in tanks (3500 l). They were kept at 10 °C in water which simulated the natural salinities of the geographic source region of the cod (KIEL — 18‰ and GDA — 8‰). The fish were maintained at natural photoperiod and acclimated to laboratory conditions for a minimum of 14 days until they start feeding and displayed typical behaviour. All experiments complied with EC Directive 2010/63/EU for animal experiments and with the permission no. 60/2012 of the Local Ethics Committee on Animal Experimentation No. 3. Samples of gills were collected and stored in RNAlater, following the manufacturer's instruction (Quiagen, Hilden, Germany). Total RNA from 36 individuals was extracted using the ISOLATE II RNA Mini Kit (Bioline, London, UK) and stored at − 20 °C. Concentration of extracted RNA was determined at 260 nm on microplate using the Epoch Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, USA). Samples were processed using a 454 pyrosequencing by CD Genomics (USA). 2.2. Transcriptome assembly
⁎ Corresponding author. Tel.: +48 58 731 17 63; fax: +48 58 551 21 30. E-mail address: [email protected] (R. Wenne).
After trimming adapters and low quality bases, a total of 962,516 reads with mean length 300–400 bp (Fig. 1) were used for de novo
http://dx.doi.org/10.1016/j.margen.2015.04.005 1874-7787/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
38
M. Małachowicz et al. / Marine Genomics 23 (2015) 37–40
2.3. Functional annotation
Fig. 1. Frequency distribution of raw reads lengths on a logarithmic scale.
transcriptome assembly using the CLC Genomics Workbench ver. 7.5.1, CLC Bio, Aarhus, Denmark (see Supplementary methods for details).
B
Of the 14,029 contigs, 100% homologous transcripts (13,585 genes) were identified using BLASTn with an E-value threshold of 10 − 10. All contigs were divided into biotypes (Fig. 2A). Gene ontology (GO) terms were assigned to each contig using the Blast2GO tool (www.blast2go.com, Götz et al., 2008). Among 13,585 genes 75.39% had an ontology definition and were classified into three main GO categories (biological process — BP, molecular function — MF and cellular component — CC) and 45 subcategories (Fig. 2B). Among these genes, 48.34% were assigned to at least one GO term in the molecular function category, 33.24% in biological processes, and 18.42% in cellular component (see Supplementary methods for details). In addition, we compared Baltic cod contigs with the Atlantic Ocean cod reference transcriptome (http://www.ensembl.org/Gadus_morhua) using BLASTn. The identified Baltic cod transcripts covered 58% of the Atlantic cod transcriptome (Fig. 3A). While 13,827 (98.56%) Baltic transcripts aligned perfectly and unambiguously, 202 (1.44%; 170 genes) demonstrated significant distinctiveness from Atlantic cod (Fig. 3B). Among those 170 genes 100% were protein coding sequences; 68.82%
A
Fig. 2. Summary of Baltic cod transcriptome assembly. (A) Among 14,029 contigs 13,704 were coding proteins, 240 were annotated as pseudogenes, and 85 as small noncoding RNAs (logarithmic scale). (B) A total of 10,242 genes were assigned to at least one GO term and were grouped into three main categories and 45 subcategories.
M. Małachowicz et al. / Marine Genomics 23 (2015) 37–40
A
39
B
C
Fig. 3. Comparison of Baltic and Atlantic cod using BLASTn and Blast2Go. (A) Baltic cod transcripts covered 58% of Atlantic cod transcriptome. (B) 1.44% (202 transcripts, 170 genes) demonstrated differences between Baltic and Atlantic cod. (C) Within these genes, 56.63% belonged to molecular function, 28.06% to biological process and 15.61% cellular component.
of them had an ontology definition and were classified into three main GO categories (BP, MF, CC) and 29 subcategories (Fig. 3C). The molecular function classes was the most highly represented (56.63%), followed by biological process (28.06%) and cellular component (15.31%) (see Supplementary methods for details). While exhibiting similarities on a broad scale, evidence of significant differences between transcriptomes from the two samples of Atlantic cod from the Baltic Sea and Atlantic cod reference was found (Fig. 3B). These differences could have been caused by geographic origin from different native waters, which confirms the need for a reference transcriptome for Baltic cod.
Acknowledgements
2.4. Nucleotide sequence accession numbers
References
The Baltic cod raw data from this study were deposited in the NCBI Sequence Read Archive under the accession number [SRP052904]. Assembled contigs and annotation information are available from the authors upon request.
This study was partially funded by project: 2011/01/M/NZ9/07207 of the National Science Centre in Poland to RW and statutory topic IV.1 in the IO PAS. We would like to thank dr. rer. nat. Ch. Petereit (GEOMAR) for fish collection from Kiel Bight, Germany. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.margen.2015.04.005.
Götz, S., Garcia-Gómez, J.M., Terol, J., Williams, T.D., Nagaraj, S.H., Nueda, M.J., Robles, M., Talón, M., Dopazo, J., Conesa, A., 2008. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res. 36, 3420–3435. http://dx. doi.org/10.1093/nar/gkn176. Lanes, C.F.C., Bizuayehu, T.T., de Oliveira Fernandes, J.M., Kiron, V., Babiak, I., 2013. Transcriptome of Atlantic Cod (Gadus morhua L.) Early embryos from farmed and wild
40
M. Małachowicz et al. / Marine Genomics 23 (2015) 37–40
broodstock. Mar. Biotechnol. 15, 677–694. http://dx.doi.org/10.1007/s10126-0139527-y. Nielsen, E.E., Hansen, M.M., Ruzzante, D.E., Meldrup, D., Grønkjær, P., 2003. Evidence of a hybrid-zone in Atlantic cod (Gadus morhua) in the Baltic and the Danish Belt Sea revealed by individual admixture analysis. Mol. Ecol. 12, 1497–1508. http://dx.doi.org/ 10.1046/j.1365-294X.2003.01819.x. Nissling, A., Westin, L., 1997. Salinity requirements for successful spawning of Baltic and Belt Sea cod and the potential for cod stock interactions in the Baltic Sea. Mar. Ecol. Prog. Ser. 152, 261–271. Nissling, A., Kryvi, H., Vallin, L., 1994. Variation in egg buoyancy of Baltic cod (Gadus morhua) and its implications for egg survival in prevailing conditions in the Baltic Sea. Mar. Ecol. Prog. Ser. 110, 67–74. O'Leary, D.B., Coughlan, J., Dillane, E., McCarthy, T.V., Cross, T.F., 2007. Microsatellite variation in cod Gadus morhua throughout its geographic range. J. Fish Biol. 70, 310–335. http://dx.doi.org/10.1111/j.1095-8649.2007.01451.x.
Poćwierz-Kotus, A., Kijewska, A., Petereit, C., Bernaś, R., Więcaszek, B., Arnyasi, M., Lien, S., Kent, M.P., Wenne, R., 2015. Genetic differentiation of brackish water populations of cod Gadus morhua in the southern Baltic, inferred from genotyping using SNP-arrays. Mar. Genomics 19, 17–22. http://dx.doi.org/10.1016/j.margen.2014.05.010. Rise, M.L., Nash, G.W., Hall, J.R., Booman, M., Hori, T.S., Trippel, E.A., Gamperl, A.K., 2014. Variation in embryonic mortality and maternal transcript expression among Atlantic cod (Gadus morhua) broodstock: a functional genomics study. Mar. Genomics 18 (Pt A), 3–20. http://dx.doi.org/10.1016/j.margen.2014.05.004. Škugor, A., Krasnov, A., Andersen, Ø., 2014. Genome-wide microarray analysis of Atlantic cod (Gadus morhua) oocyte and embryo. BMC Genomics 15, 594. http://dx.doi.org/ 10.1186/1471-2164-15-594. Star, B., Nederbragt, A.J., Jentoft, S., Grimholt, U., Malmstrom, M., Gregers, T.F., Rounge, T.B., Paulsen, J., Solbakken, M.H., Sharma, A., et al., 2011. The genome sequence of Atlantic cod reveals a unique immune system. Nature 477 (7363), 207–210. http:// dx.doi.org/10.1038/nature10342.
