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

• Published in: EvoDevo 2013-06, Vol.4 (1), Article 16
• Main Authors: Tulin, Sarah, Aguiar, Derek, Istrail, Sorin, Smith, Joel
• Format: Article
• Language: English
• Subjects: Analysis; Embryonic development; Genes; Genomes; Genomics; Pipe lines; RNA
• ISSN: 2041-9139; 2041-9139
• DOI: 10.1186/2041-9139-4-16

The de novo assembly of transcriptomes from short shotgun sequencesraises challenges due to random and non-random sequencing biases andinherent transcript complexity. We sought to define a pipeline for denovo transcriptome assembly to aid researchers working withemerging model systems where well annotated genome assemblies are notavailable as a reference. To detail this experimental and computationalmethod, we used early embryos of the sea anemone, Nematostellavectensis, an emerging model system for studies of animal body planevolution. We performed RNA-seq on embryos up to 24 h of developmentusing Illumina HiSeq technology and evaluated independent de novoassembly methods. The resulting reads were assembled using either theTrinity assembler on all quality controlled reads or both the Velvet andOases assemblers on reads passing a stringent digital normalization filter.A control set of mRNA standards from the National Institute of Standards andTechnology (NIST) was included in our experimental pipeline to invest ourtranscriptome with quantitative information on absolute transcript levelsand to provide additional quality control. We generated >200 million paired-end reads from directional cDNA librariesrepresenting well over 20 Gb of sequence. The Trinity assembler pipeline,including preliminary quality control steps, resulted in more than 86% ofreads aligning with the reference transcriptome thus generated.Nevertheless, digital normalization combined with assembly by Velvet andOases required far less computing power and decreased processing time whilestill mapping 82% of reads. We have made the raw sequencing reads andassembled transcriptome publically available. Nematostella vectensis was chosen for its strategic position in thetree of life for studies into the origins of the animal body plan, however,the challenge of reference-free transcriptome assembly is relevant to allsystems for which well annotated gene models and independently verifiedgenome assembly may not be available. To navigate this new territory, wehave constructed a pipeline for library preparation and computationalanalysis for de novo transcriptome assembly. The gene modelsdefined by this reference transcriptome define the set of genes transcribedin early Nematostella development and will provide a valuabledataset for further gene regulatory network investigations.