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The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus.

Ewen-Campen B, Shaner N, Panfilio KA, Suzuki Y, Roth S, Extavour CG - BMC Genomics (2011)

Bottom Line: We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes.We also specifically address the effects of cDNA normalization on gene discovery in de novo transcriptome analyses.Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high-throughput gene discovery for organisms lacking a sequenced genome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.

ABSTRACT

Background: Most evolutionary developmental biology ("evo-devo") studies of emerging model organisms focus on small numbers of candidate genes cloned individually using degenerate PCR. However, newly available sequencing technologies such as 454 pyrosequencing have recently begun to allow for massive gene discovery in animals without sequenced genomes. Within insects, although large volumes of sequence data are available for holometabolous insects, developmental studies of basally branching hemimetabolous insects typically suffer from low rates of gene discovery.

Results: We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug Oncopeltus fasciatus, which lacks a sequenced genome. This indirectly developing insect occupies an important phylogenetic position, branching basal to Diptera (including fruit flies) and Hymenoptera (including honeybees), and is an experimentally tractable model for short-germ development. 2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs) and 112,531 singletons. The assembled sequences fell into 16,617 unique gene models, and included predictions of splicing isoforms, which we examined experimentally. Discovery of new genes plateaued after assembly of ~1.5 million reads, suggesting that we have sequenced nearly all transcripts present in the cDNA sampled. Many transcripts have been assembled at close to full length, and there is a net gain of sequence data for over half of the pre-existing O. fasciatus accessions for developmental genes in GenBank. We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes. We also specifically address the effects of cDNA normalization on gene discovery in de novo transcriptome analyses.

Conclusions: Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high-throughput gene discovery for organisms lacking a sequenced genome. These data will have applications to the study of the evolution of arthropod genes and genetic pathways, and to the wider evolution, development and genomics communities working with emerging model organisms.[The sequence data from this study have been submitted to GenBank under study accession number SRP002610 (http://www.ncbi.nlm.nih.gov/sra?term=SRP002610). Custom scripts generated are available at http://www.extavourlab.com/protocols/index.html. Seven Additional files are available.].

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Introduction to Oncopeltus fasciatus and the workflow for producing a de novo transcriptome assembly. (A) An adult milkweed bug, Oncopeltus fasciatus. (B) Ovaries of adult female. Anterior is up. Oocytes (O) are visible in progressive stages of growth before reaching a common oviduct (Od). Oocytes are cytoplasmically connected to nurse cells (Nc) in the anterior of each ovariole. Scale bar = 1.0 mm. (C-D) The stages of O. fasciatus embryogenesis represented in this transcriptome. Embryos are stained with Sytox Green (Invitrogen) to visualize nuclei. Scale bars = 0.5 mm. (C) Development proceeds from left to right. Anterior is to the left. The cellularized blastoderm forms during the first ~20% of development (~0-24 hours at 28°C), as nuclei reach the surface of the yolk and repeatedly divide. (D) Germ band extension and segmentation occur from ~20-60% of development (~24-72 hours at 28°C). Development proceeds from left to right. Anterior is up. Mn = mandibular segment; Mx = maxillary segment; Lb = labial segment; T1-T3 = leg-bearing thoracic segments 1-3; Ab = abdomen. (E) The flow of information during this de novo transcriptome assembly project. Data files are represented as white boxes within grey boxes that indicate the computer programs used to generate these files. All of the computer programs used are freely available. Ortholog_best_hit_calculator.py and transcriptome_blast_summarizer.py are custom python scripts available at http://www.extavourlab.com/protocols/index.html (see text for details). Photograph in (A) courtesy of David Behl.
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Figure 1: Introduction to Oncopeltus fasciatus and the workflow for producing a de novo transcriptome assembly. (A) An adult milkweed bug, Oncopeltus fasciatus. (B) Ovaries of adult female. Anterior is up. Oocytes (O) are visible in progressive stages of growth before reaching a common oviduct (Od). Oocytes are cytoplasmically connected to nurse cells (Nc) in the anterior of each ovariole. Scale bar = 1.0 mm. (C-D) The stages of O. fasciatus embryogenesis represented in this transcriptome. Embryos are stained with Sytox Green (Invitrogen) to visualize nuclei. Scale bars = 0.5 mm. (C) Development proceeds from left to right. Anterior is to the left. The cellularized blastoderm forms during the first ~20% of development (~0-24 hours at 28°C), as nuclei reach the surface of the yolk and repeatedly divide. (D) Germ band extension and segmentation occur from ~20-60% of development (~24-72 hours at 28°C). Development proceeds from left to right. Anterior is up. Mn = mandibular segment; Mx = maxillary segment; Lb = labial segment; T1-T3 = leg-bearing thoracic segments 1-3; Ab = abdomen. (E) The flow of information during this de novo transcriptome assembly project. Data files are represented as white boxes within grey boxes that indicate the computer programs used to generate these files. All of the computer programs used are freely available. Ortholog_best_hit_calculator.py and transcriptome_blast_summarizer.py are custom python scripts available at http://www.extavourlab.com/protocols/index.html (see text for details). Photograph in (A) courtesy of David Behl.

Mentions: The milkweed bug Oncopeltus fasciatus (Figure 1A-D) has emerged as a promising hemipteran system for studying the molecular development of hemimetabolous insects [40-42]. It can be reared easily and cheaply in the laboratory, and has a long history as a laboratory animal for classical embryology and pattern formation studies [43-45]. More recently, robust protocols for in situ hybridization, live imaging of embryogenesis, and RNAi-mediated gene knockdown have been developed and successfully applied to the study of the evolution of development [see for example [46,47]].


The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus.

Ewen-Campen B, Shaner N, Panfilio KA, Suzuki Y, Roth S, Extavour CG - BMC Genomics (2011)

Introduction to Oncopeltus fasciatus and the workflow for producing a de novo transcriptome assembly. (A) An adult milkweed bug, Oncopeltus fasciatus. (B) Ovaries of adult female. Anterior is up. Oocytes (O) are visible in progressive stages of growth before reaching a common oviduct (Od). Oocytes are cytoplasmically connected to nurse cells (Nc) in the anterior of each ovariole. Scale bar = 1.0 mm. (C-D) The stages of O. fasciatus embryogenesis represented in this transcriptome. Embryos are stained with Sytox Green (Invitrogen) to visualize nuclei. Scale bars = 0.5 mm. (C) Development proceeds from left to right. Anterior is to the left. The cellularized blastoderm forms during the first ~20% of development (~0-24 hours at 28°C), as nuclei reach the surface of the yolk and repeatedly divide. (D) Germ band extension and segmentation occur from ~20-60% of development (~24-72 hours at 28°C). Development proceeds from left to right. Anterior is up. Mn = mandibular segment; Mx = maxillary segment; Lb = labial segment; T1-T3 = leg-bearing thoracic segments 1-3; Ab = abdomen. (E) The flow of information during this de novo transcriptome assembly project. Data files are represented as white boxes within grey boxes that indicate the computer programs used to generate these files. All of the computer programs used are freely available. Ortholog_best_hit_calculator.py and transcriptome_blast_summarizer.py are custom python scripts available at http://www.extavourlab.com/protocols/index.html (see text for details). Photograph in (A) courtesy of David Behl.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3040728&req=5

Figure 1: Introduction to Oncopeltus fasciatus and the workflow for producing a de novo transcriptome assembly. (A) An adult milkweed bug, Oncopeltus fasciatus. (B) Ovaries of adult female. Anterior is up. Oocytes (O) are visible in progressive stages of growth before reaching a common oviduct (Od). Oocytes are cytoplasmically connected to nurse cells (Nc) in the anterior of each ovariole. Scale bar = 1.0 mm. (C-D) The stages of O. fasciatus embryogenesis represented in this transcriptome. Embryos are stained with Sytox Green (Invitrogen) to visualize nuclei. Scale bars = 0.5 mm. (C) Development proceeds from left to right. Anterior is to the left. The cellularized blastoderm forms during the first ~20% of development (~0-24 hours at 28°C), as nuclei reach the surface of the yolk and repeatedly divide. (D) Germ band extension and segmentation occur from ~20-60% of development (~24-72 hours at 28°C). Development proceeds from left to right. Anterior is up. Mn = mandibular segment; Mx = maxillary segment; Lb = labial segment; T1-T3 = leg-bearing thoracic segments 1-3; Ab = abdomen. (E) The flow of information during this de novo transcriptome assembly project. Data files are represented as white boxes within grey boxes that indicate the computer programs used to generate these files. All of the computer programs used are freely available. Ortholog_best_hit_calculator.py and transcriptome_blast_summarizer.py are custom python scripts available at http://www.extavourlab.com/protocols/index.html (see text for details). Photograph in (A) courtesy of David Behl.
Mentions: The milkweed bug Oncopeltus fasciatus (Figure 1A-D) has emerged as a promising hemipteran system for studying the molecular development of hemimetabolous insects [40-42]. It can be reared easily and cheaply in the laboratory, and has a long history as a laboratory animal for classical embryology and pattern formation studies [43-45]. More recently, robust protocols for in situ hybridization, live imaging of embryogenesis, and RNAi-mediated gene knockdown have been developed and successfully applied to the study of the evolution of development [see for example [46,47]].

Bottom Line: We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes.We also specifically address the effects of cDNA normalization on gene discovery in de novo transcriptome analyses.Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high-throughput gene discovery for organisms lacking a sequenced genome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.

ABSTRACT

Background: Most evolutionary developmental biology ("evo-devo") studies of emerging model organisms focus on small numbers of candidate genes cloned individually using degenerate PCR. However, newly available sequencing technologies such as 454 pyrosequencing have recently begun to allow for massive gene discovery in animals without sequenced genomes. Within insects, although large volumes of sequence data are available for holometabolous insects, developmental studies of basally branching hemimetabolous insects typically suffer from low rates of gene discovery.

Results: We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug Oncopeltus fasciatus, which lacks a sequenced genome. This indirectly developing insect occupies an important phylogenetic position, branching basal to Diptera (including fruit flies) and Hymenoptera (including honeybees), and is an experimentally tractable model for short-germ development. 2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs) and 112,531 singletons. The assembled sequences fell into 16,617 unique gene models, and included predictions of splicing isoforms, which we examined experimentally. Discovery of new genes plateaued after assembly of ~1.5 million reads, suggesting that we have sequenced nearly all transcripts present in the cDNA sampled. Many transcripts have been assembled at close to full length, and there is a net gain of sequence data for over half of the pre-existing O. fasciatus accessions for developmental genes in GenBank. We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes. We also specifically address the effects of cDNA normalization on gene discovery in de novo transcriptome analyses.

Conclusions: Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high-throughput gene discovery for organisms lacking a sequenced genome. These data will have applications to the study of the evolution of arthropod genes and genetic pathways, and to the wider evolution, development and genomics communities working with emerging model organisms.[The sequence data from this study have been submitted to GenBank under study accession number SRP002610 (http://www.ncbi.nlm.nih.gov/sra?term=SRP002610). Custom scripts generated are available at http://www.extavourlab.com/protocols/index.html. Seven Additional files are available.].

Show MeSH
Related in: MedlinePlus