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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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Newbler 2.3 correctly identifies splicing isoforms of nanos. (A) Newbler v2.0 identified three separate contigs that map to an O. fasciatus nanos homologue that we had previously identified by degenerate PCR (Ewen-Campen & Extavour, unpublished). Newbler v2.0 failed to identify these contigs as belonging to the same transcript because of branching conflicts amongst the reads joining these contigs. BLASTX against the RefSeq protein database identified only contig 31035 as being a putative nanos homologue; the other two contigs lie outside the conserved Nanos domain and obtain no BLAST hits. (B) Newbler v2.3 predicted that the same three contigs identified by Newbler v2.0 belonged to two isotigs, or splicing isoforms. (C) RT-PCR with specific primers F and R shown in (B) resulted in two bands of the predicted sizes of the isotigs predicted by Newbler v2.3. (D) Sequencing the bands from (C) revealed that they were identical to the sequences of the predicted isotigs from (B).
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Figure 3: Newbler 2.3 correctly identifies splicing isoforms of nanos. (A) Newbler v2.0 identified three separate contigs that map to an O. fasciatus nanos homologue that we had previously identified by degenerate PCR (Ewen-Campen & Extavour, unpublished). Newbler v2.0 failed to identify these contigs as belonging to the same transcript because of branching conflicts amongst the reads joining these contigs. BLASTX against the RefSeq protein database identified only contig 31035 as being a putative nanos homologue; the other two contigs lie outside the conserved Nanos domain and obtain no BLAST hits. (B) Newbler v2.3 predicted that the same three contigs identified by Newbler v2.0 belonged to two isotigs, or splicing isoforms. (C) RT-PCR with specific primers F and R shown in (B) resulted in two bands of the predicted sizes of the isotigs predicted by Newbler v2.3. (D) Sequencing the bands from (C) revealed that they were identical to the sequences of the predicted isotigs from (B).

Mentions: To examine whether the alternative isoforms predicted by Newbler v2.3 are in fact present in developing embryos of O. fasciatus, we first focused on a gene of particular interest to developmental biologists, nanos. This conserved metazoan gene was first described as a loss of function mutation in Drosophila melanogaster [52], and is necessary for germ cell and posterior somatic development [reviewed in [53]]. Newbler v2.3 predicted this gene to encode two alternative isotigs within a single isogroup (Figure 3B). The two isotigs differ in that the longer contains an additional 100-bp exon that is absent from the shorter (Figure 3B). We designed PCR primers against sequences present in both isotigs (Figure 3B arrows), which amplified two bands differing by ~100 bp from a pool of embryonic cDNA (Figure 3C). Sequencing of these two bands confirmed that they differ exactly as predicted by Newbler v2.3 (Figure 3D).


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)

Newbler 2.3 correctly identifies splicing isoforms of nanos. (A) Newbler v2.0 identified three separate contigs that map to an O. fasciatus nanos homologue that we had previously identified by degenerate PCR (Ewen-Campen & Extavour, unpublished). Newbler v2.0 failed to identify these contigs as belonging to the same transcript because of branching conflicts amongst the reads joining these contigs. BLASTX against the RefSeq protein database identified only contig 31035 as being a putative nanos homologue; the other two contigs lie outside the conserved Nanos domain and obtain no BLAST hits. (B) Newbler v2.3 predicted that the same three contigs identified by Newbler v2.0 belonged to two isotigs, or splicing isoforms. (C) RT-PCR with specific primers F and R shown in (B) resulted in two bands of the predicted sizes of the isotigs predicted by Newbler v2.3. (D) Sequencing the bands from (C) revealed that they were identical to the sequences of the predicted isotigs from (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Newbler 2.3 correctly identifies splicing isoforms of nanos. (A) Newbler v2.0 identified three separate contigs that map to an O. fasciatus nanos homologue that we had previously identified by degenerate PCR (Ewen-Campen & Extavour, unpublished). Newbler v2.0 failed to identify these contigs as belonging to the same transcript because of branching conflicts amongst the reads joining these contigs. BLASTX against the RefSeq protein database identified only contig 31035 as being a putative nanos homologue; the other two contigs lie outside the conserved Nanos domain and obtain no BLAST hits. (B) Newbler v2.3 predicted that the same three contigs identified by Newbler v2.0 belonged to two isotigs, or splicing isoforms. (C) RT-PCR with specific primers F and R shown in (B) resulted in two bands of the predicted sizes of the isotigs predicted by Newbler v2.3. (D) Sequencing the bands from (C) revealed that they were identical to the sequences of the predicted isotigs from (B).
Mentions: To examine whether the alternative isoforms predicted by Newbler v2.3 are in fact present in developing embryos of O. fasciatus, we first focused on a gene of particular interest to developmental biologists, nanos. This conserved metazoan gene was first described as a loss of function mutation in Drosophila melanogaster [52], and is necessary for germ cell and posterior somatic development [reviewed in [53]]. Newbler v2.3 predicted this gene to encode two alternative isotigs within a single isogroup (Figure 3B). The two isotigs differ in that the longer contains an additional 100-bp exon that is absent from the shorter (Figure 3B). We designed PCR primers against sequences present in both isotigs (Figure 3B arrows), which amplified two bands differing by ~100 bp from a pool of embryonic cDNA (Figure 3C). Sequencing of these two bands confirmed that they differ exactly as predicted by Newbler v2.3 (Figure 3D).

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