Abstract
A recent study identified candidate genes linked to magnetoreception in rainbow trout (Oncorhynchus mykiss) by sequencing transcriptomes from the brains of fish exposed to a magnetic pulse. However, the discovery of these candidate genes was limited to sequences that aligned to the reference genome. The unaligned, or unmapped, sequences may yet contain valuable information resulting from regions missing, misassembled, or divergent from the reference. Using the available sequencing data from the trout brain transcriptomes, we assembled >27 million unmapped sequences (5.8% of total sequences) into 45,142 contigs and identified 12 differentially expressed contigs as a result of exposure to a pulsed magnetic field. These contigs encoded a putative superoxide dismutase – a protein necessary to prevent oxidative damage – and collagen alpha-1 type II – a structural protein important for the development and integrity of the retina. These genes were consistent with the previous study suggesting an effect of the magnetic pulse on oxidative consequences of free iron and on non-visual encephalic photoreceptors. Our results demonstrate the utility of assembling unmapped sequencing reads in studies of gene expression and identify additional candidate genes associated with a magnetic sense in trout.
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Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410. https://doi.org/10.1016/s0022-2836(05)80360-2
Anders S, McCarthy DJ, Chen Y, Okoniewski M, Smyth GK, Huber W, Robinson MD (2013) Count-based differential expression analysis of RNA sequencing data using R and Bioconductor. Nat Protoc 8(9):1765–1786. https://doi.org/10.1038/nprot.2013.099
Andrews S (2010) FastQC: a quality control tool for high throughput sequence data. Babraham Institute. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Accessed 12 Aug 2017
Antunes A, Ramos MJ (2005) Discovery of a large number of previously unrecognized mitochondrial pseudogenes in fish genomes. Genomics 86(6):708–717. https://doi.org/10.1016/j.ygeno.2005.08.002
Arniella MB, Fitak R, Johnsen S (2017) Data from: unmapped sequencing reads identify additional candidate genes linked to magnetoreception in trout. Dryad Data Repository. https://doi.org/10.5061/dryad.4g048
Audicana MT, Kennedy MW (2008) Anisakis simplex: from obscure infectious worm to inducer of immune hypersensitivity. Clin Microbiol Rev 21(2):360–379. https://doi.org/10.1128/cmr.00012-07
Barat-Houari M, Sarrabay G, Gatinois V, Fabre A, Dumont B, Genevieve D, Touitou I (2016) Mutation update for COL2A1 gene variants associated with type II collagenopathies. Hum Mutat 37(1):7–15. https://doi.org/10.1002/humu.22915
Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate - a practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol 57:289–300
Berthelot C, Brunet F, Chalopin D, Juanchich A, Bernard M, Noël B, Bento P, da Silva C, Labadie K, Alberti A, Aury JM, Louis A, Dehais P, Bardou P, Montfort J, Klopp C, Cabau C, Gaspin C, Thorgaard GH, Boussaha M, Quillet E, Guyomard R, Galiana D, Bobe J, Volff JN, Genêt C, Wincker P, Jaillon O, Crollius HR, Guiguen Y (2014) The rainbow trout genome provides novel insights into evolution after whole-genome duplication in vertebrates. Nat Commun 5:3657. https://doi.org/10.1038/ncomms4657
Blumstein DT, Berger-Tal O (2015) Understanding sensory mechanisms to develop effective conservation and management tools. Curr Opin Behav Sci 6:13–18. https://doi.org/10.1016/j.cobeha.2015.06.008
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120. https://doi.org/10.1093/bioinformatics/btu170
Carrara M, Cavallo F, Arigoni M, Calogero RA (2012) Digging in the RNA-seq garbage: evaluating the characteristics of unmapped RNA-seq reads in normal tissues. In: Sixth international conference on complex, intelligent, and software intensive systems pp 588–591. https://doi.org/10.1109/CISIS.2012.107
Chew GL, Brown GE (1989) Orientation of rainbow trout (Salmo gairdneri) in normal and null magnetic fields. Can J Zool 67(3):641–643. https://doi.org/10.1139/z89-092
Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21(18):3674–3676. https://doi.org/10.1093/bioinformatics/bti610
Conesa A, Madrigal P, Tarazona S, Gomez-Cabrero D, Cervera A, McPherson A, Szcześniak MW, Gaffney DJ, Elo LL, Zhang X, Mortazavi A (2016) A survey of best practices for RNA-seq data analysis. Genome Biol 17(1):13. https://doi.org/10.1186/s13059-016-0881-8
Cooke SJ, Sack L, Franklin CE, Farrell AP, Beardall J, Wikelski M, Chown SL (2013) What is conservation physiology? Perspectives on an increasingly integrated and essential science. Conserv Physiol 1(1):cot001. https://doi.org/10.1093/conphys/cot001
Cooke SJ, Blumstein DT, Buchholz R, Caro T, Fernández-Juricic E, Franklin CE, Metcalfe J, O’Connor CM, St. Clair CC, Sutherland WJ, Wikelski M (2014) Physiology, behavior, and conservation. Physiol Biochem Zool 87(1):1–14. https://doi.org/10.1086/671165
Deardorff TL, Kent ML (1989) Prevalence of larval Anisakis simplex in pen-reared and wild-caught salmon (Salmonidae) from Puget sound, Washington. J Wildl Dis 25(3):416–419. https://doi.org/10.7589/0090-3558-25.3.416
Deardorff TL, Kayes SG, Fukumura T (1991) Human anisakiasis transmitted by marine food products. Hawaii Med J 50(1):9–16
Denisenko-Nehrbass N, Faivre-Sarrailh C, Goutebroze L, Girault JA (2002) A molecular view on paranodal junctions of myelinated fibers. J Physiol Paris 96(1-2):99–103. https://doi.org/10.1016/S0928-4257(01)00085-7
Diebel CE, Proksch R, Green CR, Neilson P, Walker MM (2000) Magnetite defines a vertebrate magnetoreceptor. Nature 406(6793):299–302. https://doi.org/10.1038/35018561
Dobin A, Gingeras TR (2015) Mapping RNA-seq reads with STAR. Curr Protoc Bioinformatics 51:11.14.11–11.14.19. https://doi.org/10.1002/0471250953.bi1114s51
Edelman NB, Fritz T, Nimpf S, Pichler P, Lauwers M, Hickman RW, Papadaki-Anastasopoulou A, Ushakova L, Heuser T, Resch GP, Saunders M, Shaw JA, Keays DA (2015) No evidence for intracellular magnetite in putative vertebrate magnetoreceptors identified by magnetic screening. Proc Natl Acad Sci U S A 112(1):262–267. https://doi.org/10.1073/pnas.1407915112
Eder SHK, Cadiou H, Muhamad A, McNaughton PA, Kirschvink JL, Winklhofer M (2012) Magnetic characterization of isolated candidate vertebrate magnetoreceptor cells. Proc Natl Acad Sci U S A 109(30):12022–12027. https://doi.org/10.1073/pnas.1205653109
Faber-Hammond JJ, Brown KH (2016) Pseudo-de novo assembly and analysis of unmapped genome sequence reads in wild zebrafish reveal novel gene content. Zebrafish 13(2):95–102. https://doi.org/10.1089/zeb.2015.1154
Fitak RR, Wheeler BR, Ernst DA, Lohmann KJ, Johnsen S (2017) Candidate genes mediating magnetoreception in rainbow trout (Oncorhynchus mykiss). Biol Lett 13(4):20170142. https://doi.org/10.1098/rsbl.2017.0142
Gadadhar S, Bodakuntla S, Natarajan K, Janke C (2017) The tubulin code at a glance. J Cell Sci 130(8):1347–1353. https://doi.org/10.1242/jcs.199471
Gouin A, Legeai F, Nouhaud P, Whibley A, Simon JC, Lemaitre C (2015) Whole-genome re-sequencing of non-model organisms: lessons from unmapped reads. Heredity 114(5):494–501. https://doi.org/10.1038/hdy.2014.85
Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, 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(7):644–652. https://doi.org/10.1038/nbt.1883
Grotwinkel JT, Wild K, Segnitz B, Sinning I (2014) SRP RNA remodeling by SRP68 explains its role in protein translocation. Science 344(6179):101–104. https://doi.org/10.1126/science.1249094
Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, MacManes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, LeDuc RD, Friedman N, Regev A (2013) De novo transcript sequence reconstruction from RNA-seq using the trinity platform for reference generation and analysis. Nat Protoc 8(8):1494–1512. https://doi.org/10.1038/nprot.2013.084
Hellinger J, Hoffmann KP (2009) Magnetic field perception in the rainbow trout, Oncorhynchus mykiss. J Comp Physiol A 195(9):873–879. https://doi.org/10.1007/s00359-009-0466-z
Hellinger J, Hoffmann KP (2012) Magnetic field perception in the rainbow trout Oncorynchus mykiss: magnetite mediated, light dependent or both? J Comp Physiol A 198(8):593–605. https://doi.org/10.1007/s00359-012-0732-3
Hsu CY, Ko FY, Li CW, Fann K, Lue JT (2007) Magnetoreception system in honeybees (Apis mellifera). PLoS One 2(4):e395. https://doi.org/10.1371/journal.pone.0000395
Johnsen S, Lohmann KJ (2005) The physics and neurobiology of magnetoreception. Nat Rev Neurosci 6(9):703–712. https://doi.org/10.1038/nrn1745
Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, McWilliam H, Maslen J, Mitchell A, Nuka G, Pesseat S, Quinn AF, Sangrador-Vegas A, Scheremetjew M, Yong SY, Lopez R, Hunter S (2014) InterProScan 5: genome-scale protein function classification. Bioinformatics 30(9):1236–1240. https://doi.org/10.1093/bioinformatics/btu031
Knight M, Ongele E, Lewis FA (2000) Molecular studies of Biomphalaria glabrata, an intermediate host of Schistosoma mansoni. Int J Parasitol 30(4):535–541. https://doi.org/10.1016/S0020-7519(99)00182-4
Langmead B, Salzberg SL (2012) Fast gapped-read alignment with bowtie 2. Nat Methods 9(4):357–359. https://doi.org/10.1038/nmeth.1923
Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12(1):323. https://doi.org/10.1186/1471-2105-12-323
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079. https://doi.org/10.1093/bioinformatics/btp352
Liu YW, Zhou J, White KP (2014) RNA-seq differential expression studies: more sequence or more replication? Bioinformatics 30(3):301–304. https://doi.org/10.1093/bioinformatics/btt688
Margalit A, Brachner A, Gotzmann J, Foisner R, Gruenbaum Y (2007) Barrier-to-autointegration factor--a BAFfling little protein. Trends Cell Biol 17(4):202–208. https://doi.org/10.1016/j.tcb.2007.02.004
Mattick JS, Rinn JL (2015) Discovery and annotation of long noncoding RNAs. Nat Struct Mol Biol 22(1):5–7. https://doi.org/10.1038/nsmb.2942
McCarthy DJ, Chen Y, Smyth GK (2012) Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res 40(10):4288–4297. https://doi.org/10.1093/nar/gks042
Putman NF, Lohmann KJ, Putman EM, Quinn TP, Klimley AP, Noakes DLG (2013) Evidence for geomagnetic imprinting as a homing mechanism in Pacific salmon. Curr Biol 23(4):312–316. https://doi.org/10.1016/j.cub.2012.12.041
Putman NF, Scanlan MM, Billman EJ, O’Neil JP, Couture RB, Quinn TP, Lohmann KJ, DLG N (2014a) An inherited magnetic map guides ocean navigation in juvenile Pacific salmon. Curr Biol 24(4):446–450. https://doi.org/10.1016/j.cub.2014.01.017
Putman NF, Jenkins ES, Michielsens CGJ, Noakes DLG (2014b) Geomagnetic imprinting predicts spatio-temporal variation in homing migration of pink and sockeye salmon. J R Soc Interface 11(99):10. https://doi.org/10.1098/rsif.2014.0542
Putman NF, Meinke AM, Noakes DL (2014c) Rearing in a distorted magnetic field disrupts the 'map sense' of juvenile steelhead trout. Biol Lett 10(6):20140169. https://doi.org/10.1098/rsbl.2014.0169
Reinert K, Langmead B, Weese D, Evers DJ (2015) Alignment of next-generation sequencing reads. Annu Rev Genomics Hum Genet 16(1):133–151. https://doi.org/10.1146/annurev-genom-090413-025358
Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26(1):139–140. https://doi.org/10.1093/bioinformatics/btp616
Rondeau EB, Minkley DR, Leong JS, Messmer AM, Jantzen JR, von Schalburg KR, Lemon C, Bird NH, Koop BF (2014) The genome and linkage map of the northern pike (Esox lucius): conserved synteny revealed between the salmonid sister group and the Neoteleostei. PLoS One 9(7):e102089. https://doi.org/10.1371/journal.pone.0102089
Shaw J, Boyd A, House M, Woodward R, Mathes F, Cowin G, Saunders M, Baer B (2015) Magnetic particle-mediated magnetoreception. J R Soc Interface 12(110):20150499. https://doi.org/10.1098/rsif.2015.0499
Sheng Y, Abreu IA, Cabelli DE, Maroney MJ, Miller AF, Teixeira M, Valentine JS (2014) Superoxide dismutases and superoxide reductases. Chem Rev 114(7):3854–3918. https://doi.org/10.1021/cr4005296
Sleptsova-Friedrich I, Li Y, Emelyanov A, Ekker M, Korzh V, Ge R (2001) Fgfr3 and regionalization of anterior neural tube in zebrafish. Mech Dev 102(1-2):213–217. https://doi.org/10.1016/S0925-4773(01)00280-5
Smith-Unna R, Boursnell C, Patro R, Hibberd JM, Kelly S (2016) TransRate: reference-free quality assessment of de novo transcriptome assemblies. Genome Res 26(8):1134–1144. https://doi.org/10.1101/gr.196469.115
Tran-Viet KN, Soler V, Quiette V, Powell C, Yanovitch T, Metlapally R, Luo X, Katsanis N, Nading E, Young TL (2013) Mutation in collagen II alpha 1 isoforms delineates stickler and Wagner syndrome phenotypes. Mol Vis 19:759–766
Usman T, Hadlich F, Demasius W, Weikard R, Kuhn C (2017) Unmapped reads from cattle RNAseq data: a source for missing and misassembled sequences in the reference assemblies and for detection of pathogens in the host. Genomics 109(1):36–42. https://doi.org/10.1016/j.ygeno.2016.11.009
Walbaum JJ (1792) Petri Artedi sueci genera piscium in quibus systema totum ichthyologiae proponitur cum classibus, ordinibus, generum characteribus, specierum differentiis, observationibus plurimis: redactis speciebus 242 ad genera 52: Ichthyologiae pars 3. Impensis Ant. Ferdin. Röse, Grypeswaldiae.
Walker MM, Diebel CE, Haugh CV, Pankhurst PM, Montgomery JC, Green CR (1997) Structure and function of the vertebrate magnetic sense. Nature 390(6658):371–376. https://doi.org/10.1038/37057
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10(1):57–63. https://doi.org/10.1038/nrg2484
Whitacre LK, Tizioto PC, Kim JW, Sonstegard TS, Schroeder SG, Alexander LJ, Medrano JF, Schnabel RD, Taylor JF, Decker JE (2015) What's in your next-generation sequence data? An exploration of unmapped DNA and RNA sequence reads from the bovine reference individual. BMC Genomics 16(1):1114. https://doi.org/10.1186/s12864-015-2313-7
Williams AG, Thomas S, Wyman SK, Holloway AK (2014) RNA-seq data: challenges in and recommendations for experimental design and analysis. Curr Protoc Hum Genet 83:11.13.11–11.13.20. https://doi.org/10.1002/0471142905.hg1113s83
Wood AW, Matsumoto J, Van Der Kraak G (2004) Thyroglobulin type-1 domain protease inhibitors exhibit specific expression in the cortical ooplasm of vitellogenic rainbow trout oocytes. Mol Reprod Dev 69(2):205–214. https://doi.org/10.1002/mrd.20118
Zhu W, Wang L, Dong Z, Chen X, Song F, Liu N, Yang H, Fu J (2016) Comparative transcriptome analysis identifies candidate genes related to skin color differentiation in red tilapia. Sci Rep 6(1):31347. https://doi.org/10.1038/srep31347
Acknowledgements
We would like to thank the Duke Shared Cluster Resource for providing the computational resources necessary for the project. We also thank E. Caves, L. Schweikert, and J. Notar for comments on earlier drafts of this manuscript. This work was supported by the Duke University Scholars Program [to M.B.A.] and the Air Force Office of Scientific Research [FA9550-14-1-0208 to S.J. and R.R.F].
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Arniella, M.B., Fitak, R.R. & Johnsen, S. Unmapped sequencing reads identify additional candidate genes linked to magnetoreception in rainbow trout. Environ Biol Fish 101, 711–721 (2018). https://doi.org/10.1007/s10641-018-0731-5
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DOI: https://doi.org/10.1007/s10641-018-0731-5