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

Abstract

BackgroundThe 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 de novo 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, Nematostella vectensis, 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.ResultsWe 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.ConclusionsNematostella 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.

Gene expression profiling during the embryo‐to‐larva transition in the giant red sea urchin Mesocentrotus franciscanus

“…However , thanks to the advances in next‐generation sequencing (NGS) technologies, the ability to sequence the entire transcriptome of a given tissue or life‐history stage in a matter of days is providing new opportunities to explore the complexity of developmental GRNs in other echinoderms (Delroisse, Ortega‐Martinez, Dupont, Mallefet, & Flammang, 2015; Dilly, Gaitán‐Espitia, & Hofmann, 2015; Dylus et al., 2016; Gildor, Malik, Sher, Avraham, & Ben‐Tabou de‐Leon, 2015; Tulin, Aguiar, Istrail, & Smith, 2013) and marine invertebrates (Jackson & Degnan, 2016; Layden, Rentzsch, & Röttinger, 2016). As changes in gene expression may underlie many of the phenotypic differences between species (Brawand et al., 2011), studying transcriptomic divergence of sympatric species may shed light upon the initial genetic targets of natural selection in speciation events (Filteau, Pavey, St‐Cyr, & Bernatchez, 2013).…”

Current directions and future perspectives from the third Nematostella research conference

“…Two recently published studies have characterized transcriptomic changes during Nematostella development (Helm et al, 2013; Tulin et al, 2013). In addition, several studies were presented on transcriptomic profiles in Nematostella associated with particular biological processes, tissues, or structures.…”

“…This approach has been extensively applied in characterizing embryonic development of several model species including fruit fly (Jaeger et al, 2004; Aerts et al, 2010), sea urchin (Smith et al, 2007), and zebrafish (Chan et al, 2009; Greenhill et al, 2011). Given the relatively simple body plan and cell division pattern during Nematostella embryogenesis, the availability of a relatively large amount of gene expression data, and the potential to manipulate gene expression levels through morpholino knockdown and overexpression, Nematostella provides a tractable species for computational modeling of development (Helm et al, 2013; Tulin et al, 2013). Jaap Kaandorp’s laboratory (University of Amsterdam, Netherlands) has developed methods to quantitatively analyze images from in situ hybridization studies conducted in Nematostella (Botman and Kaandorp, 2012).…”

Using Nematostella vectensis to Study the Interactions between Genome, Epigenome, and Bacteria in a Changing Environment

“…It is easily cultured in laboratory in high numbers Uhlinger, 1992, 1995) and clonally propagated to eliminate genetic confounding effects. In addition, N. vectensis has the unique advantage amongst marine invertebrates of having extensive sequencing of transcriptomes (Helm et al, 2013;Tulin et al, 2013), preexisting data on genome methylation (Zemach et al, 2010), histone modifications (Schwaiger et al, 2014), and miRNAs (Moran et al, 2014). These existing data result in an exceptionally well-annotated genome, an essential but still rare tool for marine species.…”