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  • Transcriptome  (2)
  • BioMed Central  (2)
  • American Institute of Physics (AIP)
  • Public Library of Science (PLoS)
  • 2010-2014  (2)
  • 1
    Publication Date: 2022-05-25
    Description: © The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in EvoDevo 4 (2013): 16, doi:10.1186/2041-9139-4-16.
    Description: 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 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. 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. 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.
    Keywords: Transcriptome ; Gene regulatory network ; Nematostella embryonic development ; Body plan evolution ; Next-generation sequencing ; Illumina HiSeq ; Trinity ; Oases ; RNA-seq
    Repository Name: Woods Hole Open Access Server
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  • 2
    Publication Date: 2022-05-26
    Description: © The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Genomics 14 (2013): 266, doi:10.1186/1471-2164-14-266.
    Description: Nematostella vectensis, a burrowing sea anemone, has become a popular species for the study of cnidarian development. In previous studies, the expression of a variety of genes has been characterized during N. vectensis development with in situ mRNA hybridization. This has provided detailed spatial resolution and a qualitative perspective on changes in expression. However, little is known about broad transcriptome-level patterns of gene expression through time. Here we examine the expression of N. vectensis genes through the course of development with quantitative RNA-seq. We provide an overview of changes in the transcriptome through development, and examine the maternal to zygotic transition, which has been difficult to investigate with other tools. We measured transcript abundance in N. vectensis with RNA-seq at six time points in development: zygote (2 hours post fertilization (HPF)), early blastula (7 HPF), mid-blastula (12 HPF), gastrula (24 HPF), planula (5 days post fertilization (DPF)) and young polyp (10 DPF). The major wave of zygotic expression appears between 7–12 HPF, though some changes occur between 2–7 HPF. The most dynamic changes in transcript abundance occur between the late blastula and early gastrula stages. More transcripts are upregulated between the gastrula and planula than downregulated, and a comparatively lower number of transcripts significantly change between planula and polyp. Within the maternal to zygotic transition, we identified a subset of maternal factors that decrease early in development, and likely play a role in suppressing zygotic gene expression. Among the first genes to be expressed zygotically are genes whose proteins may be involved in the degradation of maternal RNA. The approach presented here is highly complementary to prior studies on spatial patterns of gene expression, as it provides a quantitative perspective on a broad set of genes through time but lacks spatial resolution. In addition to addressing the problems identified above, our work provides an annotated matrix that other investigators can use to examine genes and developmental events that we do not examine in detail here.
    Description: This work was supported by seed funds from the Brown-MBL Partnership and the National Science Foundation Graduate Student Research Fellowship. Infrastructure for data transfer from the sequencer was supported by the National Science Foundation EPSCoR Program under Grant No. 1004057 (Infrastructure to Advance Life Sciences in the Ocean State).
    Keywords: Nematostella vectensis ; Transcriptome ; Gene expression ; Maternal to zygotic transition ; Development
    Repository Name: Woods Hole Open Access Server
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