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Identification of xenobiotic biodegradation and metabolism-related genes in the copepod Tigriopus japonicus whole transcriptome analysis
MARGEN-00333; No of Pages 2 Marine Genomics xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Genomics/Technical resources
Identification of xenobiotic biodegradation and metabolism-related genes in the copepod Tigriopus japonicus whole transcriptome analysis Hui-Su Kim a,1, Bo-Young Lee a,1, Eun-Ji Won a, Jeonghoon Han a, Dae-Sik Hwang a, Heum Gi Park b, Jae-Seong Lee a,⁎ a b
Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 440-746, South Korea Department of Marine Resource Development, College of Life Sciences, Gangneung-Wonju National University, Gangneung 210-702, South Korea
a r t i c l e
i n f o
Article history: Received 11 May 2015 Received in revised form 15 May 2015 Accepted 15 May 2015 Available online xxxx Keywords: Whole transcriptome analysis Environmental stimulus Copepod Tigriopus japonicus
a b s t r a c t In this study, the whole transcriptome of Tigriopus japonicus was sequenced using next generation sequencing technology. De novo assembly was performed using Trinity, which assembled 140,130 contigs. Transdecoder found 54,761 candidate coding contigs, 39,507 of which showed homology to other species covering 15,310 genes by BLAST analysis. Functional gene annotation was performed by Gene Ontology, InterProScan, and KEGG pathway analyses. In addition to various metabolism-related pathways, xenobiotic biodegradation and metabolism were other interesting pathways in T. japonicus. Transcripts encoding various enzymes (e.g. superoxide dismutase, heat shock protein, and peroxidases) in response to a variety of stimuli were identified, which might be useful candidate biomarkers for ecotoxicology studies. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Currently, the Arthropod Genomic Consortium has launched the i5k project, which will sequence the genomes of 5000 arthropod species including copepods (https://i5k.nal.usda.gov/). Copepod is one of the targeted taxa that have been ranked as a model species for this project. In fact, copepod is the second largest taxa of crustacean and is dominant in aquatic zooplankton communities accounting for about 70% of the biomass of marine zooplankton (Wells, 1984). The intertidal benthic copepod Tigriopus japonicus inhabits rock pools and is distributed along the coasts of the Western Pacific with wide ranges of adaptability for salinity and temperature (Kwok and Leung, 2005). Their benthic life habit means this species meets the requirement for monitoring the sediment-water interface (Raisuddin et al., 2007). Thus, ecology, phylogeography, and behavior studies have been conducted for monitoring the environmental stressors and pollution in diverse marine environments in the genus Tigriopus (Kwok and Leung, 2005). Toxicity tests using different chemicals, including trace metals, endocrinedisrupting chemicals (EDC), and engineered nanomaterials have been performed in T. japonicus (Lee et al., 2007).
⁎ Corresponding author. Tel.: +82 31 290 7011. E-mail address: [email protected] (J.-S. Lee). 1 These authors contributed equally to this work.
To date, fragmentary knowledge on molecular defense mechanisms has been increasingly reported from the genus Tigriopus, and T. japonicus is considered a promising new model species for ecotoxicology (Raisuddin et al., 2007). Genome and transcriptome sequences of Tigriopus califonicus are now available via the i5k project (https://i5k. nal.usda.gov/Tigriopus_californicus), increasing the usefulness of T. japonicus in view of parallel studies. Through parallel studies of the genus Tigriopus, valuable comparative information will be provided that can be used to examine molecular mechanisms and the evolution of sister species within the genus Tigriopus. 2. Data description 2.1. Sequence assembly and gene annotation After sequencing, sequence reads with a Q-score below 30 and a length shorter than 90 bp were removed for quality control, and the remaining sequences were used for further analysis. The large contigs of the sequence reads, filtered by the quality control, were constructed using the de novo assembler Trinity (Grabherr et al., 2011). TransDecoder (http://transdecoder.sourceforge.net/) was used to identify candidate coding regions from the assembled transcripts and/ or contigs; the candidate coding regions were used for BLAST analysis against the NCBI non-redundant (nr) protein database. The presence of conserved domains in the assembled transcripts was identified and
http://dx.doi.org/10.1016/j.margen.2015.05.011 1874-7787/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Kim, H.-S., et al., Identification of xenobiotic biodegradation and metabolism-related genes in the copepod Tigriopus japonicus whole transcriptome analysis, Mar. Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.05.011
2
H.-S. Kim et al. / Marine Genomics xxx (2015) xxx–xxx
annotated using InterProScan5 (Zdobnov and Apweiler, 2001). Gene Ontology and KEGG pathway analysis of the contigs were performed using Blast2GO (Conesa et al., 2005). 2.2. De novo assembly of T. japonicus transcript and identification of coding sequences Sequencing and assembly results are summarized in Table 1. Using the sequence reads that passed quality control, we assembled 140,130 contigs with Trinity with a size range of 201 to 30,174 bp. Among the assembled contigs, TransDecoder found 54,761 contigs that contain candidate coding regions (Table 1). Overall size distribution of the final contigs is shown in Supplementary file S1. Among 54,761 final contigs filtered by TransDecoder, 5444 contigs (9.94%) were multiple open reading frames (ORFs) from a single transcript. The sequences of the transcript contigs were deposited to the Transcriptome Shotgun Assembly (TSA) database in GenBank (Accession no. GCHA01000000). 2.3. Annotation of genes and protein domains BLAST analysis found that 39,507 contigs (72%) had positive matches (e-value b1e-06) to homologous genes of other species, which represents a potential of 15,310 genes (Supplementary file S1). The sequence distribution of the top BLAST hit species, with more than 400 hits, is shown in Supplementary file S2. InterProScan5 detected putative protein domains from 37,666 contig sequences, 27,706 of which were annotated by GO terms (Supplementary file S3) (Boschetti et al., 2012).
Table 1 Sequence reads and contigs. Sequence analysis
Counts
Length (bp)
Raw sequence reads acquired Sequence reads after QC Contigs after trinity assembly Size range of contigs Average length Median length N50 Contigs after TransDecoder Size range of contigs Average length Median length
108,072,846 94,223,440 140,130
9,486,783,007 9,371,373,254 237,625,532 201 to 30,174 1695 780 3565 82,984,914 297 to 23,769 1515 1038
54,761
xenobiotic biodegradation and metabolism: aminobenzoate degradation, steroid degradation, drug metabolism-cytochrome P450, drug metabolism-other enzymes, metabolism of xenobiotics by cytochrome P450, caprolactam degradation, toluene degradation, and styrene degradation. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.margen.2015.05.011. Acknowledgments This work was supported by a grant from the Marine Biotechnology Program (PJT200620, Genome analysis of marine organisms and development of functional application) funded by the Ministry of Oceans and Fisheries, Korea, to Jae-Seong Lee.
2.4. Gene ontology and KEGG pathway analysis References Gene Ontology (GO) and KEGG pathway analysis allow for the functional annotation of T. japonicus transcript sequences. All the results of GO analysis are summarized at the second level, and GO terms related to the top domains are described in Supplementary file S1. Analysis of KEGG pathway showed that most annotated sequences were related to metabolism pathways, including amino acid metabolism, nucleotide metabolism, glycan biosynthesis and metabolism, carbohydrate metabolism, lipid metabolism, energy metabolism, metabolism of cofactors and vitamins, xenobiotic biodegradation, biosynthesis of other secondary metabolites, and metabolism of terpenoids and polyketides, while only 7.3% of annotated sequences were involved in the pathways of signal transduction, translation, and the immune system (Supplementary files S4, S5). 2.5. Xenobiotic biodegradation and metabolism Among the 13 pathways identified by KEGG analysis from the T. japanicus transcripts, 52 sequences were related to xenobiotic biodegradation and metabolism (SupplementaryfilesS2, S3). The KEGG pathways analysis showed eight different pathways that belong to
Boschetti, C., Carr, A., Crisp, A., Eyres, I., Wang-Koh, Y., Lubzens, E., Barraclough, T.G., Micklem, G., Tunnacliffe, A., 2012. Biochemical diversification through foreign gene expression in Bdelloid Rotifers. PLoS Genet. 8, e1003035. Conesa, A., Gotz, S., Garcia-Gomez, J.M., Terol, J., Talon, M., Robles, M., 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, 3674–3676. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B.W., Nusbaum, C., Lindblad-Toh, K., Friedman, N., Regev, A., 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–652. Kwok, K.W., Leung, K.M., 2005. Toxicity of antifouling biocides to the intertidal harpacticoid copepod Tigriopus japonicus (Crustacea, Copepoda): effects of temperature and salinity. Mar. Pollut. Bull. 51, 830–837. Lee, K.-W., Raisuddin, S., Hwang, D.-S., Park, H.G., Lee, J.-S., 2007. Acute toxicities of trace metals and common xenobiotics to the marine copepod Tigriopus japonicus: evaluation of its use as a benchmark species for routine ecotoxicity tests in Western Pacific coastal regions. Environ. Toxicol. 22, 532–538. Raisuddin, S., Kwok, K.W., Leung, K.M., Schlenk, D., Lee, J.-S., 2007. The copepod Tigriopus: a promising marine model organism for ecotoxicology and environmental genomics. Aquat. Toxicol. 83, 161–173. Wells, P.G., 1984. Ecotoxicological Testing for the Marine Environment. In: Persoone, G., Jaspers, E., Claus, C. (Eds.), Inst. Mar. Sci. Res.Springer, Bredene, pp. 215–256 Zdobnov, E.M., Apweiler, R., 2001. InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17, 847–848.
Please cite this article as: Kim, H.-S., et al., Identification of xenobiotic biodegradation and metabolism-related genes in the copepod Tigriopus japonicus whole transcriptome analysis, Mar. Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.05.011
Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Genomics/Technical resources
Identification of xenobiotic biodegradation and metabolism-related genes in the copepod Tigriopus japonicus whole transcriptome analysis Hui-Su Kim a,1, Bo-Young Lee a,1, Eun-Ji Won a, Jeonghoon Han a, Dae-Sik Hwang a, Heum Gi Park b, Jae-Seong Lee a,⁎ a b
Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 440-746, South Korea Department of Marine Resource Development, College of Life Sciences, Gangneung-Wonju National University, Gangneung 210-702, South Korea
a r t i c l e
i n f o
Article history: Received 11 May 2015 Received in revised form 15 May 2015 Accepted 15 May 2015 Available online xxxx Keywords: Whole transcriptome analysis Environmental stimulus Copepod Tigriopus japonicus
a b s t r a c t In this study, the whole transcriptome of Tigriopus japonicus was sequenced using next generation sequencing technology. De novo assembly was performed using Trinity, which assembled 140,130 contigs. Transdecoder found 54,761 candidate coding contigs, 39,507 of which showed homology to other species covering 15,310 genes by BLAST analysis. Functional gene annotation was performed by Gene Ontology, InterProScan, and KEGG pathway analyses. In addition to various metabolism-related pathways, xenobiotic biodegradation and metabolism were other interesting pathways in T. japonicus. Transcripts encoding various enzymes (e.g. superoxide dismutase, heat shock protein, and peroxidases) in response to a variety of stimuli were identified, which might be useful candidate biomarkers for ecotoxicology studies. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Currently, the Arthropod Genomic Consortium has launched the i5k project, which will sequence the genomes of 5000 arthropod species including copepods (https://i5k.nal.usda.gov/). Copepod is one of the targeted taxa that have been ranked as a model species for this project. In fact, copepod is the second largest taxa of crustacean and is dominant in aquatic zooplankton communities accounting for about 70% of the biomass of marine zooplankton (Wells, 1984). The intertidal benthic copepod Tigriopus japonicus inhabits rock pools and is distributed along the coasts of the Western Pacific with wide ranges of adaptability for salinity and temperature (Kwok and Leung, 2005). Their benthic life habit means this species meets the requirement for monitoring the sediment-water interface (Raisuddin et al., 2007). Thus, ecology, phylogeography, and behavior studies have been conducted for monitoring the environmental stressors and pollution in diverse marine environments in the genus Tigriopus (Kwok and Leung, 2005). Toxicity tests using different chemicals, including trace metals, endocrinedisrupting chemicals (EDC), and engineered nanomaterials have been performed in T. japonicus (Lee et al., 2007).
⁎ Corresponding author. Tel.: +82 31 290 7011. E-mail address: [email protected] (J.-S. Lee). 1 These authors contributed equally to this work.
To date, fragmentary knowledge on molecular defense mechanisms has been increasingly reported from the genus Tigriopus, and T. japonicus is considered a promising new model species for ecotoxicology (Raisuddin et al., 2007). Genome and transcriptome sequences of Tigriopus califonicus are now available via the i5k project (https://i5k. nal.usda.gov/Tigriopus_californicus), increasing the usefulness of T. japonicus in view of parallel studies. Through parallel studies of the genus Tigriopus, valuable comparative information will be provided that can be used to examine molecular mechanisms and the evolution of sister species within the genus Tigriopus. 2. Data description 2.1. Sequence assembly and gene annotation After sequencing, sequence reads with a Q-score below 30 and a length shorter than 90 bp were removed for quality control, and the remaining sequences were used for further analysis. The large contigs of the sequence reads, filtered by the quality control, were constructed using the de novo assembler Trinity (Grabherr et al., 2011). TransDecoder (http://transdecoder.sourceforge.net/) was used to identify candidate coding regions from the assembled transcripts and/ or contigs; the candidate coding regions were used for BLAST analysis against the NCBI non-redundant (nr) protein database. The presence of conserved domains in the assembled transcripts was identified and
http://dx.doi.org/10.1016/j.margen.2015.05.011 1874-7787/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Kim, H.-S., et al., Identification of xenobiotic biodegradation and metabolism-related genes in the copepod Tigriopus japonicus whole transcriptome analysis, Mar. Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.05.011
2
H.-S. Kim et al. / Marine Genomics xxx (2015) xxx–xxx
annotated using InterProScan5 (Zdobnov and Apweiler, 2001). Gene Ontology and KEGG pathway analysis of the contigs were performed using Blast2GO (Conesa et al., 2005). 2.2. De novo assembly of T. japonicus transcript and identification of coding sequences Sequencing and assembly results are summarized in Table 1. Using the sequence reads that passed quality control, we assembled 140,130 contigs with Trinity with a size range of 201 to 30,174 bp. Among the assembled contigs, TransDecoder found 54,761 contigs that contain candidate coding regions (Table 1). Overall size distribution of the final contigs is shown in Supplementary file S1. Among 54,761 final contigs filtered by TransDecoder, 5444 contigs (9.94%) were multiple open reading frames (ORFs) from a single transcript. The sequences of the transcript contigs were deposited to the Transcriptome Shotgun Assembly (TSA) database in GenBank (Accession no. GCHA01000000). 2.3. Annotation of genes and protein domains BLAST analysis found that 39,507 contigs (72%) had positive matches (e-value b1e-06) to homologous genes of other species, which represents a potential of 15,310 genes (Supplementary file S1). The sequence distribution of the top BLAST hit species, with more than 400 hits, is shown in Supplementary file S2. InterProScan5 detected putative protein domains from 37,666 contig sequences, 27,706 of which were annotated by GO terms (Supplementary file S3) (Boschetti et al., 2012).
Table 1 Sequence reads and contigs. Sequence analysis
Counts
Length (bp)
Raw sequence reads acquired Sequence reads after QC Contigs after trinity assembly Size range of contigs Average length Median length N50 Contigs after TransDecoder Size range of contigs Average length Median length
108,072,846 94,223,440 140,130
9,486,783,007 9,371,373,254 237,625,532 201 to 30,174 1695 780 3565 82,984,914 297 to 23,769 1515 1038
54,761
xenobiotic biodegradation and metabolism: aminobenzoate degradation, steroid degradation, drug metabolism-cytochrome P450, drug metabolism-other enzymes, metabolism of xenobiotics by cytochrome P450, caprolactam degradation, toluene degradation, and styrene degradation. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.margen.2015.05.011. Acknowledgments This work was supported by a grant from the Marine Biotechnology Program (PJT200620, Genome analysis of marine organisms and development of functional application) funded by the Ministry of Oceans and Fisheries, Korea, to Jae-Seong Lee.
2.4. Gene ontology and KEGG pathway analysis References Gene Ontology (GO) and KEGG pathway analysis allow for the functional annotation of T. japonicus transcript sequences. All the results of GO analysis are summarized at the second level, and GO terms related to the top domains are described in Supplementary file S1. Analysis of KEGG pathway showed that most annotated sequences were related to metabolism pathways, including amino acid metabolism, nucleotide metabolism, glycan biosynthesis and metabolism, carbohydrate metabolism, lipid metabolism, energy metabolism, metabolism of cofactors and vitamins, xenobiotic biodegradation, biosynthesis of other secondary metabolites, and metabolism of terpenoids and polyketides, while only 7.3% of annotated sequences were involved in the pathways of signal transduction, translation, and the immune system (Supplementary files S4, S5). 2.5. Xenobiotic biodegradation and metabolism Among the 13 pathways identified by KEGG analysis from the T. japanicus transcripts, 52 sequences were related to xenobiotic biodegradation and metabolism (SupplementaryfilesS2, S3). The KEGG pathways analysis showed eight different pathways that belong to
Boschetti, C., Carr, A., Crisp, A., Eyres, I., Wang-Koh, Y., Lubzens, E., Barraclough, T.G., Micklem, G., Tunnacliffe, A., 2012. Biochemical diversification through foreign gene expression in Bdelloid Rotifers. PLoS Genet. 8, e1003035. Conesa, A., Gotz, S., Garcia-Gomez, J.M., Terol, J., Talon, M., Robles, M., 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, 3674–3676. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B.W., Nusbaum, C., Lindblad-Toh, K., Friedman, N., Regev, A., 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–652. Kwok, K.W., Leung, K.M., 2005. Toxicity of antifouling biocides to the intertidal harpacticoid copepod Tigriopus japonicus (Crustacea, Copepoda): effects of temperature and salinity. Mar. Pollut. Bull. 51, 830–837. Lee, K.-W., Raisuddin, S., Hwang, D.-S., Park, H.G., Lee, J.-S., 2007. Acute toxicities of trace metals and common xenobiotics to the marine copepod Tigriopus japonicus: evaluation of its use as a benchmark species for routine ecotoxicity tests in Western Pacific coastal regions. Environ. Toxicol. 22, 532–538. Raisuddin, S., Kwok, K.W., Leung, K.M., Schlenk, D., Lee, J.-S., 2007. The copepod Tigriopus: a promising marine model organism for ecotoxicology and environmental genomics. Aquat. Toxicol. 83, 161–173. Wells, P.G., 1984. Ecotoxicological Testing for the Marine Environment. In: Persoone, G., Jaspers, E., Claus, C. (Eds.), Inst. Mar. Sci. Res.Springer, Bredene, pp. 215–256 Zdobnov, E.M., Apweiler, R., 2001. InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17, 847–848.
Please cite this article as: Kim, H.-S., et al., Identification of xenobiotic biodegradation and metabolism-related genes in the copepod Tigriopus japonicus whole transcriptome analysis, Mar. Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.05.011