Gene discovery using massively parallel pyrosequencing to develop ESTs for the flesh fly Sarcophaga crassipalpis.
Gene discovery using massively parallel pyrosequencing to develop ESTs for the flesh fly Sarcophaga crassipalpis.
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DOI：
10.1186/1471-2164-10-234
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发表时间：
2009-05-19
期刊：
影响因子：
4.4
通讯作者：
Denlinger DL
中科院分区：
文献类型：
--
作者：
Hahn DA;Ragland GJ;Shoemaker DD;Denlinger DL
肉蝇属是研究内分泌、滞育、抗寒、生殖和免疫等方面的重要模型。尽管肉蝇作为昆虫生理学和生物化学模型的突出地位，以及在法医学研究中，很少有该属成员的基因组或转录组数据。我们在Roche 454-FLX平台上使用大规模并行焦磷酸测序来产生肉蝇Sarcophaga crassipis的大量EST数据集。为了最大限度地提高序列多样性，我们汇集了从所有生命阶段的全身提取的RNA，并在逆转录后将cDNA池标准化。我们获得了207，110个EST，平均阅读长度为241 bp。这些读段组装成20，995个重叠群和31，056个单例。使用NR和NT数据库的BLAST搜索，我们能够鉴定11，757个独特的基因元件（E<0.0001），代表大约9，000个独立的转录物。比较了S.在GO生物过程功能组中，crassipalpis unigenes的EST与果蝇转录组的EST的比较表明，我们的EST广泛地代表了肉蝇转录组。454测序中的插入和缺失错误是比较转录组分析的一个严重障碍。借助于一种新的方法来纠正这些错误，我们进行了比较分析的遗传分歧之间的GO类之间的S。crassipalpis、毛须藓D.黑腹按蚊和冈比亚按蚊。结果表明，非同义替换发生在不同类别的相似率，虽然相关的基因对刺激的反应可能会进化得稍微快一点。此外，我们在EST中鉴定了500多个潜在的微卫星位点和12，000多个SNP。我们的数据为肉蝇提供了第一个大规模的EST项目，这是探索这种模式物种所急需的资源。此外，我们发现了大量潜在的微卫星和SNP标记，可用于群体和系统研究的S。crassipalpis和其他肉蝇。
Flesh flies in the genus Sarcophaga are important models for investigating endocrinology, diapause, cold hardiness, reproduction, and immunity. Despite the prominence of Sarcophaga flesh flies as models for insect physiology and biochemistry, and in forensic studies, little genomic or transcriptomic data are available for members of this genus. We used massively parallel pyrosequencing on the Roche 454-FLX platform to produce a substantial EST dataset for the flesh fly Sarcophaga crassipalpis. To maximize sequence diversity, we pooled RNA extracted from whole bodies of all life stages and normalized the cDNA pool after reverse transcription. We obtained 207,110 ESTs with an average read length of 241 bp. These reads assembled into 20,995 contigs and 31,056 singletons. Using BLAST searches of the NR and NT databases we were able to identify 11,757 unique gene elements (E<0.0001) representing approximately 9,000 independent transcripts. Comparison of the distribution of S. crassipalpis unigenes among GO Biological Process functional groups with that of the Drosophila melanogaster transcriptome suggests that our ESTs are broadly representative of the flesh fly transcriptome. Insertion and deletion errors in 454 sequencing present a serious hurdle to comparative transcriptome analysis. Aided by a new approach to correcting for these errors, we performed a comparative analysis of genetic divergence across GO categories among S. crassipalpis, D. melanogaster, and Anopheles gambiae. The results suggest that non-synonymous substitutions occur at similar rates across categories, although genes related to response to stimuli may evolve slightly faster. In addition, we identified over 500 potential microsatellite loci and more than 12,000 SNPs among our ESTs. Our data provides the first large-scale EST-project for flesh flies, a much-needed resource for exploring this model species. In addition, we identified a large number of potential microsatellite and SNP markers that could be used in population and systematic studies of S. crassipalpis and other flesh flies.
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影响因子：
12.3
作者：
Huse SM;Huber JA;Morrison HG;Sogin ML;Welch DM
通讯作者：
Welch DM
影响因子：
56.9
作者：
LEE, RE;CHEN, CP;DENLINGER, DL
通讯作者：
DENLINGER, DL
影响因子：
2.9
作者：
Natori, S;Shiraishi, H;Kobayashi, A
通讯作者：
Kobayashi, A
DOI：
10.1073/pnas.0703538104
发表时间：
2007-07-03
影响因子：
11.1
作者：
Rinehart, Joseph P.;Li, Aiqing;Denlinger, David L.
通讯作者：
Denlinger, David L.
影响因子：
7.7
作者：
Faircloth, Brant C.
通讯作者：
Faircloth, Brant C.