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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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Assessing coverage of the O. fasciatus transcriptome. Randomly chosen subsets of increasing numbers of Titanium reads were used to generate progressively larger sub-assemblies. The number of reads in each sub-assembly (X axis) is plotted against the number of unique BLAST hits in each sub-assembly (left Y axis: black), and against the N50 isotig length (right Y axis: grey). For this analysis BLAST was performed against the SwissProt database. The number of unique BLAST hits plateaus when the assembly is composed of approximately 1.5 million reads. However, the N50 isotig length maintains an approximately constant rate of increase.
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Figure 5: Assessing coverage of the O. fasciatus transcriptome. Randomly chosen subsets of increasing numbers of Titanium reads were used to generate progressively larger sub-assemblies. The number of reads in each sub-assembly (X axis) is plotted against the number of unique BLAST hits in each sub-assembly (left Y axis: black), and against the N50 isotig length (right Y axis: grey). For this analysis BLAST was performed against the SwissProt database. The number of unique BLAST hits plateaus when the assembly is composed of approximately 1.5 million reads. However, the N50 isotig length maintains an approximately constant rate of increase.

Mentions: To address the first question, we created eight separate assemblies of progressively larger sub-samples of our total reads and tallied the total number of genes identified via BLASTX. The number of newly discovered genes began to plateau after ~1.5M reads (1 7/8 plates in our case) (Figure 5 black line). However, the N50 isotig length continued to increase roughly linearly over this range of reads (Figure 5 grey line). These results suggest that additional sequencing of this sample is unlikely to identify substantially more genes, but may continue to lengthen the existing sequences. Although in the absence of a sequenced genome it is not possible to accurately estimate how many genes are in fact present in the O. fasciatus transcriptome, we note that while several developmental genes of interest were identified in this study, others were not. (Tables 3, 4 and see below). Because these data suggest that we have sequenced these specific cDNA samples quite deeply, some form of specific target enrichment may be necessary for future attempts to discover additional genes not identified in this dataset.


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)

Assessing coverage of the O. fasciatus transcriptome. Randomly chosen subsets of increasing numbers of Titanium reads were used to generate progressively larger sub-assemblies. The number of reads in each sub-assembly (X axis) is plotted against the number of unique BLAST hits in each sub-assembly (left Y axis: black), and against the N50 isotig length (right Y axis: grey). For this analysis BLAST was performed against the SwissProt database. The number of unique BLAST hits plateaus when the assembly is composed of approximately 1.5 million reads. However, the N50 isotig length maintains an approximately constant rate of increase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Assessing coverage of the O. fasciatus transcriptome. Randomly chosen subsets of increasing numbers of Titanium reads were used to generate progressively larger sub-assemblies. The number of reads in each sub-assembly (X axis) is plotted against the number of unique BLAST hits in each sub-assembly (left Y axis: black), and against the N50 isotig length (right Y axis: grey). For this analysis BLAST was performed against the SwissProt database. The number of unique BLAST hits plateaus when the assembly is composed of approximately 1.5 million reads. However, the N50 isotig length maintains an approximately constant rate of increase.
Mentions: To address the first question, we created eight separate assemblies of progressively larger sub-samples of our total reads and tallied the total number of genes identified via BLASTX. The number of newly discovered genes began to plateau after ~1.5M reads (1 7/8 plates in our case) (Figure 5 black line). However, the N50 isotig length continued to increase roughly linearly over this range of reads (Figure 5 grey line). These results suggest that additional sequencing of this sample is unlikely to identify substantially more genes, but may continue to lengthen the existing sequences. Although in the absence of a sequenced genome it is not possible to accurately estimate how many genes are in fact present in the O. fasciatus transcriptome, we note that while several developmental genes of interest were identified in this study, others were not. (Tables 3, 4 and see below). Because these data suggest that we have sequenced these specific cDNA samples quite deeply, some form of specific target enrichment may be necessary for future attempts to discover additional genes not identified in this dataset.

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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Related in: MedlinePlus