Limits...
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.].

Show MeSH

Related in: MedlinePlus

Ortholog hit ratio analysis of isotigs and CAP3-reassembled singletons. An ortholog hit ratio of one implies that a transcript has been assembled to its true full length. For isotigs (black), a majority (54.8%) appear to contain at least 50% of the full length transcript sequence (arrow), while over one-third (37.2%) appear to represent at least 80% of the full length transcript sequence (arrowhead). Most singletons (grey) represent much smaller percentages of full-length transcripts.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Ortholog hit ratio analysis of isotigs and CAP3-reassembled singletons. An ortholog hit ratio of one implies that a transcript has been assembled to its true full length. For isotigs (black), a majority (54.8%) appear to contain at least 50% of the full length transcript sequence (arrow), while over one-third (37.2%) appear to represent at least 80% of the full length transcript sequence (arrowhead). Most singletons (grey) represent much smaller percentages of full-length transcripts.

Mentions: To address the second question, we employed a method proposed by O'Neil and colleagues [20] for addressing the question of how closely our sequences approached full-length transcripts. Their metric, the "ortholog hit ratio," compares the length of the newly discovered sequence that obtains a BLAST hit versus the full length of its top hit [20]. Thus, an ortholog hit ratio of one implies that a transcript has been assembled to its true full length, while values over one suggest insertions in the query sequence relative to its top BLAST hit. We note the caveat that many genes contain relatively poorly conserved regions that may fail to obtain a BLAST hit at all, causing the ortholog hit ratio to be an underestimate in these cases (Additional file 5). In our dataset, many of the O. fasciatus isotigs appear to be nearly fully assembled, while the singletons predictably tend to represent small portions of their top BLAST hit in RefSeq (Figure 6). In total, of the 7,219 isotigs with BLAST hits, 3,953 (54.8%) had ratios > 0.5 and 2,689 (37.2%) had ratios > 0.8.


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)

Ortholog hit ratio analysis of isotigs and CAP3-reassembled singletons. An ortholog hit ratio of one implies that a transcript has been assembled to its true full length. For isotigs (black), a majority (54.8%) appear to contain at least 50% of the full length transcript sequence (arrow), while over one-third (37.2%) appear to represent at least 80% of the full length transcript sequence (arrowhead). Most singletons (grey) represent much smaller percentages of full-length transcripts.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Ortholog hit ratio analysis of isotigs and CAP3-reassembled singletons. An ortholog hit ratio of one implies that a transcript has been assembled to its true full length. For isotigs (black), a majority (54.8%) appear to contain at least 50% of the full length transcript sequence (arrow), while over one-third (37.2%) appear to represent at least 80% of the full length transcript sequence (arrowhead). Most singletons (grey) represent much smaller percentages of full-length transcripts.
Mentions: To address the second question, we employed a method proposed by O'Neil and colleagues [20] for addressing the question of how closely our sequences approached full-length transcripts. Their metric, the "ortholog hit ratio," compares the length of the newly discovered sequence that obtains a BLAST hit versus the full length of its top hit [20]. Thus, an ortholog hit ratio of one implies that a transcript has been assembled to its true full length, while values over one suggest insertions in the query sequence relative to its top BLAST hit. We note the caveat that many genes contain relatively poorly conserved regions that may fail to obtain a BLAST hit at all, causing the ortholog hit ratio to be an underestimate in these cases (Additional file 5). In our dataset, many of the O. fasciatus isotigs appear to be nearly fully assembled, while the singletons predictably tend to represent small portions of their top BLAST hit in RefSeq (Figure 6). In total, of the 7,219 isotigs with BLAST hits, 3,953 (54.8%) had ratios > 0.5 and 2,689 (37.2%) had ratios > 0.8.

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