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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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GO term distribution of BLAST hits from the O. fasciatus transcriptome compared with those from the D. melanogaster genome. Several GO categories are shown within the top-level divisions of Biological Process, Molecular Function, and Cellular Component. Column heights reflect the percentage of annotated sequences in each assembly that mapped to a given Biological Process GO term. The relative percentages of genes falling into GO categories are comparable between our O. fasciatus transcriptome (black) and the D. melanogaster transcriptome (white).
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Figure 4: GO term distribution of BLAST hits from the O. fasciatus transcriptome compared with those from the D. melanogaster genome. Several GO categories are shown within the top-level divisions of Biological Process, Molecular Function, and Cellular Component. Column heights reflect the percentage of annotated sequences in each assembly that mapped to a given Biological Process GO term. The relative percentages of genes falling into GO categories are comparable between our O. fasciatus transcriptome (black) and the D. melanogaster transcriptome (white).

Mentions: To explore and summarize the functional categories of the genes sequenced in this study, we obtained the Gene Ontology (GO) terms associated with the top 20 BLAST hits of each sequence using Blast2GO [54]. Among the 7,059 genes for which we obtained GO terms, we observed a wide diversity of functional categories represented on all levels of the Gene Ontology database (Figure 4). The O. fasciatus sequences fall into GO categories with a roughly similar distribution to that of the well-annotated Drosophila melanogaster genome, suggesting that our sequence data contain a large diversity of genes involved in a variety of biological processes, and do not contain any notable biases towards particular categories of genes.


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)

GO term distribution of BLAST hits from the O. fasciatus transcriptome compared with those from the D. melanogaster genome. Several GO categories are shown within the top-level divisions of Biological Process, Molecular Function, and Cellular Component. Column heights reflect the percentage of annotated sequences in each assembly that mapped to a given Biological Process GO term. The relative percentages of genes falling into GO categories are comparable between our O. fasciatus transcriptome (black) and the D. melanogaster transcriptome (white).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040728&req=5

Figure 4: GO term distribution of BLAST hits from the O. fasciatus transcriptome compared with those from the D. melanogaster genome. Several GO categories are shown within the top-level divisions of Biological Process, Molecular Function, and Cellular Component. Column heights reflect the percentage of annotated sequences in each assembly that mapped to a given Biological Process GO term. The relative percentages of genes falling into GO categories are comparable between our O. fasciatus transcriptome (black) and the D. melanogaster transcriptome (white).
Mentions: To explore and summarize the functional categories of the genes sequenced in this study, we obtained the Gene Ontology (GO) terms associated with the top 20 BLAST hits of each sequence using Blast2GO [54]. Among the 7,059 genes for which we obtained GO terms, we observed a wide diversity of functional categories represented on all levels of the Gene Ontology database (Figure 4). The O. fasciatus sequences fall into GO categories with a roughly similar distribution to that of the well-annotated Drosophila melanogaster genome, suggesting that our sequence data contain a large diversity of genes involved in a variety of biological processes, and do not contain any notable biases towards particular categories of genes.

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