The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus.
The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus.
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DOI：
10.1186/1471-2164-12-61
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发表时间：
2011-01-25
期刊：
影响因子：
4.4
通讯作者：
Extavour CG
中科院分区：
文献类型：
--
作者：
Ewen-Campen B;Shaner N;Panfilio KA;Suzuki Y;Roth S;Extavour CG
大多数新兴模式生物的进化发育生物学（“evo-devo”）研究集中在使用简并PCR单独克隆的少量候选基因上。然而，最近可用的测序技术，如454焦磷酸测序，已经开始允许在没有测序基因组的动物中进行大规模基因发现。在昆虫中，虽然大量的序列数据可用于全变态昆虫，发育研究的基部分支hemimetabolous昆虫通常遭受基因发现率低。我们使用454焦磷酸测序对来自马利筋虫Oncopeltus fasciatus的卵巢和胚胎的超过5亿个碱基的cDNA进行测序，该虫缺乏测序的基因组。这种间接发育的昆虫占据了重要的系统发育位置，在双翅目（包括果蝇）和膜翅目（包括蜜蜂）的基础上分支，并且是短胚发育的实验易处理模型。来自标准化和非标准化cDNA的2，087，410个读段组装成21，097个序列（isotig）和112，531个单例。组装的序列落入16，617个独特的基因模型中，并包括剪接异构体的预测，我们通过实验进行了检查。新基因的发现在组装~ 150万个读段后趋于稳定，这表明我们已经对样品cDNA中存在的几乎所有转录本进行了测序。许多转录本已经被组装成接近全长的，并且对于超过一半的预先存在的O.在GenBank中检索发育相关基因。我们鉴定了10，775个独特的基因，包括所有主要保守的后生动物信号通路的成员和参与几个主要类别的早期发育过程的基因。我们还特别强调了cDNA标准化对从头转录组分析中基因发现的影响。我们的测序、组装和注释框架提供了一种简单有效的方法，可以为缺乏测序基因组的生物体实现高通量基因发现。这些数据将应用于节肢动物基因和遗传途径的进化研究，以及与新兴模式生物合作的更广泛的进化，发展和基因组学社区。[The来自该研究的序列数据已提交给GenBank，研究登录号为SRP 002610（http：www.ncbi.nlm.nih.gov/sra? term= SRP 002610）。生成的自定义脚本可在http://www.extavourlab.com/protocols/index.html上获得。七个附加文件可用。]
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. 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. 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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