A quantitative reference transcriptome for Nematostella vectensis earlyembryonic development: a pipeline for de novo assembly in emergingmodel systems

EvoDevo

Joel Smith²  Sorin Istrail¹  Derek Aguiar¹  Sarah Tulin² 

[1] Department of Computer Science and Center for Computational MolecularBiology, Brown University, 115 Waterman Street, Box 1910, Providence, RI02912, USA;Eugene Bell Center for Regenerative Biology and Tissue Engineering, MarineBiological Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA

关键词: RNA-seq;    Oases;    Trinity;    Illumina HiSeq;    Next-generation sequencing;    Body plan evolution;    Nematostella embryonic development;    Gene regulatory network;    Transcriptome;   

Others  :  806628
DOI  :  10.1186/2041-9139-4-16

 received in 2013-01-04, accepted in 2013-03-14,  发布年份 2013

【 摘 要 】

Background

The de novo assembly of transcriptomes from short shotgun sequences raises challenges due to random and non-random sequencing biases and inherent transcript complexity. We sought to define a pipeline for denovo transcriptome assembly to aid researchers working with emerging model systems where well annotated genome assemblies are not available as a reference. To detail this experimental and computational method, we used early embryos of the sea anemone, Nematostellavectensis, an emerging model system for studies of animal body plan evolution. We performed RNA-seq on embryos up to 24 h of development using Illumina HiSeq technology and evaluated independent de novo assembly methods. The resulting reads were assembled using either the Trinity assembler on all quality controlled reads or both the Velvet and Oases assemblers on reads passing a stringent digital normalization filter. A control set of mRNA standards from the National Institute of Standards and Technology (NIST) was included in our experimental pipeline to invest our transcriptome with quantitative information on absolute transcript levels and to provide additional quality control.

Results

We generated >200 million paired-end reads from directional cDNA libraries representing well over 20 Gb of sequence. The Trinity assembler pipeline, including preliminary quality control steps, resulted in more than 86% of reads aligning with the reference transcriptome thus generated. Nevertheless, digital normalization combined with assembly by Velvet and Oases required far less computing power and decreased processing time while still mapping 82% of reads. We have made the raw sequencing reads and assembled transcriptome publically available.

Conclusions

Nematostella vectensis was chosen for its strategic position in the tree of life for studies into the origins of the animal body plan, however, the challenge of reference-free transcriptome assembly is relevant to all systems for which well annotated gene models and independently verified genome assembly may not be available. To navigate this new territory, we have constructed a pipeline for library preparation and computational analysis for de novo transcriptome assembly. The gene models defined by this reference transcriptome define the set of genes transcribed in early Nematostella development and will provide a valuable dataset for further gene regulatory network investigations.

【 授权许可】

   
2013 Tulin et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, Smith SA, Seaver E, Rouse GW, Obst M, Edgecombe GD, Sørensen MV, Haddock SHD, Schmidt-Rhaesa A, Okusu A, Kristensen RM, Wheeler WC, Martindale MQ, Giribet G: Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 2008, 452:745-749.
• [2]Hejnol A, Obst M, Stamatakis A, Ott M, Rouse GW, Edgecombe GD, Martinez P, Baguna J, Bailly X, Jondelius U, Wiens M, Muller WEG, Seaver E, Wheeler WC, Martindale MQ, Giribet G, Dunn CW: Assessing the root of bilaterian animals with scalable phylogenomic methods. Proc R Soc B 2009, 276:4261-4270.
• [3]Mallatt J, Craig CW, Yoder MJ: Nearly complete rRNA genes assembled from across the metazoan animals: effects of more taxa, a structure-based alignment, and paired-sites evolutionary models on phylogeny reconstruction. Mol Phylogenet Evol 2010, 55:1-17.
• [4]Martindale MQ, Pang K, Finnerty JR: Investigating the origins of triploblasty: “mesodermal” gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum, Cnidaria; class, Anthozoa). Development 2004, 131:2463-2474.
• [5]Wikramanayake AH, Hong M, Lee PN, Pang K, Byrum CA, Bince JM, Xu R, Martindale MQ: An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation. Nature 2003, 426:446-450.
• [6]Fritzenwanker JH, Saina M, Technau U: Analysis of forkhead and snail expression reveals epithelial-mesenchymal transitions during embryonic and larval development of Nematostella vectensis. Dev Biol 2004, 275:389-402.
• [7]Matus DQ, Thomsen GH, Martindale MQ: Dorso/ventral genes are asymmetrically expressed and involved in germ-layer demarcation during cnidarian gastrulation. Curr Biol 2006, 16:499-505.
• [8]Rentzsch F, Anton R, Saina M, Hammerschmidt M, Holstein TW, Technau U: Asymmetric expression of the BMP antagonists chordin and gremlin in the sea anemone Nematostella vectensis: implications for the evolution of axial patterning. Dev Biol 2006, 296:375-387.
• [9]Stathopoulos A, Levine M: Genomic regulatory networks and animal development. Dev Cell 2005, 9:449-462.
• [10]Davidson EH, Levine MS: Properties of developmental gene regulatory networks. Proc Natl Acad Sci USA 2008, 105:20063-20066.
• [11]Davidson EH: The Regulatory Genome. London, UK: Academic Press; 2006.
• [12]Imai KS, Levine M, Satoh N, Satou Y: Regulatory blueprint for a chordate embryo. Science 2006, 312:1183-1187.
• [13]Betancur P, Bronner-Fraser M, Sauka-Spengler T: Assembling neural crest regulatory circuits into a gene regulatory network. Annu Rev Cell Dev Biol 2010, 26:581-603.
• [14]Swiers G, Patient R, Loose M: Genetic regulatory networks programming hematopoietic stem cells and erythroid lineage specification. Dev Biol 2006, 294:525-540.
• [15]Tu Q, Cameron RA, Worley KC, Gibbs RA, Davidson EH: Gene structure in the sea urchin Strongylocentrotus purpuratus based on transcriptome analysis. Genome Res 2012, 22:2079-2087.
• [16]Martin J, Wang Z: Next-generation transcriptome assembly. Nat Rev Genet 2011, 12:671-682.
• [17]Brown CT, Howe A, Zhang Q, Pyrkosz A, Brom T: A reference-free algorithm for computational normalization of shotgun sequencing data. [http://arxiv.org/abs/1203.4802 webcite]
• [18]Miller JR, Koren S, Sutton G: Assembly algorithms for next-generation sequencing data. Genomics 2010, 95:315-327.
• [19]Salzberg SL, Yorke JA: Beware of mis-assembled genomes. Bioinformatics 2005, 21:4320-4321.
• [20]Ryan JF, Burton PM, Mazza ME, Kwong GK, Mullikin JC, Finnerty JR: The cnidarian-bilaterian ancestor possessed at least 56 homeoboxes: evidence from the starlet sea anemone, Nematostella vectensis. Genome Biol 2006, 7:R64. BioMed Central Full Text
• [21]Putnam NH, Srivastava M, Hellsten U, Dirks B, Chapman J, Salamov A, Terry A, Shapiro H, Lindquist E, Kapitonov VV, Jurka J, Genikhovich G, Grigoriev IV, Lucas SM, Steele RE, Finnerty JR, Technau U, Martindale MQ, Rokhsar DS: Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 2007, 317:86-94.
• [22]Zeng V, Villanueva KE, Ewen-Campen BS, Alwes F, Browne WE, Extavour CG: De novo assembly and characterization of a maternal and developmental transcriptome for the emerging model crustacean Parhyale hawaiensis. BMC Genomics 2011, 12:581. BioMed Central Full Text
• [23]Ewen-Campen B, Shaner N, Panfilio KA, Suzuki Y, Roth S, Extavour CG: The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus. BMC Genomics 2011, 12:61. BioMed Central Full Text
• [24]Conaco C, Neveu P, Zhou H, Arcila ML, Degnan SM, Degnan BM, Kosik KS: Transcriptome profiling of the demosponge Amphimedon queenslandica reveals genome-wide events that accompany major life cycle transitions. BMC Genomics 2012, 13:209. BioMed Central Full Text
• [25]Robertson G, Schein J, Chiu R, Corbett R, Field M, Jackman SD, Mungall K, Lee S, Okada HM, Qian JQ, Griffith M, Raymond A, Thiessen N, Cezard T, Butterfield YS, Newsome R, Chan SK, She R, Varhol R, Kamoh B, Prabhu A-L, Tam A, Zhao Y, Moore RA, Hirst M, Marra MA, Jones SJM, Hoodless PA, Birol I: De novo assembly and analysis of RNA-seq data. Nat Methods 2010, 7:909-912.
• [26]Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, Li Y, Li S, Shan G, Kristiansen K, Li S, Yang H, Wang J, Wang J: De novo assembly of human genomes with massively parallel short read sequencing. Genome Res 2010, 20:265-272.
• [27]Zerbino DR, Birney E: Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008, 18:821-829.
• [28]Schulz MH, Zerbino DR, Vingron M, Birney E: Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels. Bioinformatics 2012, 28:1086-1092.
• [29]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: Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat Biotechnol 2011, 29:644-652.
• [30]Zhao Q-Y, Wang Y, Kong Y-M, Luo D, Li X, Hao P: Optimizing de novo transcriptome assembly from short-read RNA-seq data: a comparative study. BMC Bioinformatics 2011, 12(Suppl 14):S2. BioMed Central Full Text
• [31]Hansen KD, Brenner SE, Dudoit S: Biases in Illumina transcriptome sequencing caused by random hexamer priming. Nucleic Acids Res 2010, 38:e131-e131.
• [32]Venables WN, Ripley BD: Modern Applied Statistics with S. Berlin, Germany: Springer Verlag; 2002.
• [33]Ren X, Liu T, Dong J, Sun L, Yang J, Zhu Y, Jin Q: Evaluating de bruijn graph assemblers on 454 transcriptomic data. PLoS ONE 2012, 7:e51188.
• [34]Vijay N, Poelstra JW, Künstner A, Wolf JBW: Challenges and strategies in transcriptome assembly and differential gene expression quantification. A comprehensive in silico assessment of RNA-seq experiments. Mol Ecol 2013, 22:620-634.
• [35]Baker SC, Bauer SR, Beyer RP, Brenton JD, Bromley B, Burrill J, Causton H, Conley MP, Elespuru R, Fero M, Foy C, Fuscoe J, Gao X, Gerhold DL, Gilles P, Goodsaid F, Guo X, Hackett J, Hockett RD, Ikonomi P, Irizarry RA, Kawasaki ES, Kaysser-Kranich T, Kerr K, Kiser G, Koch WH, Lee KY, Liu C, Liu ZL, Lucas A, et al.: The External RNA Controls Consortium: a progress report. Nat Methods 2005, 2:731-734.
• [36]Devonshire AS, Elaswarapu R, Foy CA: Evaluation of external RNA controls for the standardisation of gene expression biomarker measurements. BMC Genomics 2010, 11:662. BioMed Central Full Text
• [37]External RNA Controls Consortium: Proposed methods for testing and selecting the ERCC external RNA controls. BMC Genomics 2005, 6:150.
• [38]Li B, Dewey CN: RSEM: accurate transcript quantification from RNA-seq data with or without a reference genome. BMC Bioinformatics 2011, 12:323. BioMed Central Full Text
• [39]Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M: Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21:3674-3676.
• [40]Alexa A, Rahnenfuher J: topGO: enrichment analysis for gene ontology. [http://www.bioconductor.org/packages/2.11/bioc/html/topGO.html webcite]
• [41]Marlow H, Roettinger E, Boekhout M, Martindale MQ: Functional roles of Notch signaling in the cnidarian Nematostella vectensis. Dev Biol 2012, 362:295-308.
• [42]Röttinger E, Dahlin P, Martindale MQ: A framework for the establishment of a Cnidarian gene regulatory network for “endomesoderm” specification: the inputs of β-catenin/TCF signaling. PLoS Genet 2012, 8:e1003164.
• [43]Tarazona S, García-Alcalde F, Dopazo J, Ferrer A, Conesa A: Differential expression in RNA-seq: a matter of depth. Genome Res 2011, 21:2213-2223.
• [44]Yamada A, Pang K, Martindale MQ, Tochinai S: Surprisingly complex T-box gene complement in diploblastic metazoans. Evol Dev 2007, 9:220-230.
• [45]Adamska M, Larroux C, Adamski M, Green K, Lovas E, Koop D, Richards GS, Zwafink C, Degnan BM: Structure and expression of conserved Wnt pathway components in the demosponge Amphimedon queenslandica. Evol Dev 2010, 12:494-518.
• [46]Giresi PG: Chromatin profiles of human cells in health and disease using FAIRE. Ann Arbor, MI: ProQuest; 2012.