Guerrero F D, Dowd S E, Djikeng A, Wiley G, Macmil S, Saldivar L, Najar F, Roe B A
USDA-ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory; 2700 Fredericksburg Rd., Kerrville, TX 78028 , USA.
J Med Entomol. 2009 Sep;46(5):1109-16. doi: 10.1603/033.046.0518.
We used an expressed sequence tag and 454 pyrosequencing approach to initiate a study of the genome of the screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae). Two normalized cDNA libraries were constructed from RNA isolated from embryos and second instar larvae from the Panama 95 strain. Approximately 5,400 clones from each library were sequenced from both the 5' and 3' directions using the Sanger method. In addition, double-stranded cDNA was prepared from random-primed polyA RNA purified from embryos, second-instar larvae, adult males, and adult females. These four cDNA samples were used for 454 pyrosequencing that produced approximately 300,000 independent sequences. Sequences were assembled into a database of assembled contigs and singletons and used to search public protein databases and annotate the sequences. The full database consists of 6,076 contigs and 58,221 singletons assembled from both the traditional expressed sequence tag (EST) and 454 sequences. Annotation of the data led to the identification of several gene coding regions with possible roles in sex determination in the screwworm. This database will facilitate the design of microarray and other experiments to study screwworm gene expression on a larger scale than previously possible.
我们采用表达序列标签和454焦磷酸测序方法，启动了一项关于螺旋锥蝇（Cochliomyia hominivorax (Coquerel)，双翅目：丽蝇科）基因组的研究。从巴拿马95品系的胚胎和二龄幼虫中分离出RNA，构建了两个标准化的cDNA文库。使用桑格法从每个文库中大约5400个克隆的5'和3'方向进行测序。此外，从胚胎、二龄幼虫、成年雄性和成年雌性中纯化的随机引物polyA RNA制备双链cDNA。这四个cDNA样本用于454焦磷酸测序，产生了大约300,000个独立序列。序列被组装成一个由重叠群和单拷贝组成的数据库，并用于搜索公共蛋白质数据库和注释序列。完整的数据库由从传统表达序列标签（EST）和454序列组装而成的6076个重叠群和58221个单拷贝组成。对数据的注释导致鉴定出几个可能在螺旋锥蝇性别决定中起作用的基因编码区域。该数据库将有助于设计微阵列和其他实验，以比以前更大规模地研究螺旋锥蝇的基因表达。
