Next-generation pyrosequencing of gonad transcriptomes in the polyploid lake sturgeon (Acipenser fulvescens): the relative merits of normalization and rarefaction in gene discovery.
Next-generation pyrosequencing of gonad transcriptomes in the polyploid lake sturgeon (Acipenser fulvescens): the relative merits of normalization and rarefaction in gene discovery.
复制标题
DOI：
10.1186/1471-2164-10-203
复制
发表时间：
2009-04-29
期刊：
影响因子：
4.4
通讯作者：
Dewoody JA
中科院分区：
文献类型：
--
作者：
Hale MC;McCormick CR;Jackson JR;Dewoody JA
下一代测序技术最常应用于与模型密切相关的模型生物或物种。然而，这些方法在许多野生生物中具有潜在的价值，包括那些受保护的生物。我们使用罗氏454焦磷酸测序来表征多倍体湖鲟鱼（Acipenser fulvescens）性腺的基因表达。在Roche 454 GS-FLX上进行的滴定运行产生了超过47，000个测序读数。这些读段代表通过质量控制的20，741个独特序列（平均长度= 186 bp）。这些被组装成1，831个重叠群（平均重叠群深度= 4.1个序列）。超过4，000个测序读数（约19%）被分配基因本体，主要是蛋白质，RNA和离子结合。从> 50个不同的基因中鉴定了总共877个候选SNP。我们采用了理论生态学（稀疏）的分析方法来评估相对于基因发现的测序覆盖深度。我们还考虑了使用下一代测序平台时归一化与天然cDNA文库的相对优点。毫不奇怪，标准化文库中的rRNA亚基基因较少。稀疏性表明，标准化对基因发现的效率几乎没有影响，至少在处理来自单一组织类型的数千个读数时是如此。我们的数据表明，在454个测序仪上进行滴定可以表征数千个表达的序列标签，这些序列标签可用于识别SNP、基因本体和保护物种中的基因表达水平。我们预计，稀疏将是有用的基因发现的评估和下一代测序技术具有很大的潜力，为其他非模式生物的研究。
Next-generation sequencing technologies have been applied most often to model organisms or species closely related to a model. However, these methods have the potential to be valuable in many wild organisms, including those of conservation concern. We used Roche 454 pyrosequencing to characterize gene expression in polyploid lake sturgeon (Acipenser fulvescens) gonads. Titration runs on a Roche 454 GS-FLX produced more than 47,000 sequencing reads. These reads represented 20,741 unique sequences that passed quality control (mean length = 186 bp). These were assembled into 1,831 contigs (mean contig depth = 4.1 sequences). Over 4,000 sequencing reads (~19%) were assigned gene ontologies, mostly to protein, RNA, and ion binding. A total of 877 candidate SNPs were identified from > 50 different genes. We employed an analytical approach from theoretical ecology (rarefaction) to evaluate depth of sequencing coverage relative to gene discovery. We also considered the relative merits of normalized versus native cDNA libraries when using next-generation sequencing platforms. Not surprisingly, fewer genes from the normalized libraries were rRNA subunits. Rarefaction suggests that normalization has little influence on the efficiency of gene discovery, at least when working with thousands of reads from a single tissue type. Our data indicate that titration runs on 454 sequencers can characterize thousands of expressed sequence tags which can be used to identify SNPs, gene ontologies, and levels of gene expression in species of conservation concern. We anticipate that rarefaction will be useful in evaluations of gene discovery and that next-generation sequencing technologies hold great potential for the study of other non-model organisms.
登录
查看更多内容
影响因子：
3.7
作者：
Ospina-Alvarez N;Piferrer F
通讯作者：
Piferrer F
影响因子：
0.9
作者：
Jackson, JR;VanDeValk, AJ;Rudstam, LG
通讯作者：
Rudstam, LG
影响因子：
0.9
作者：
McCormick, C. R.;Bos, D. H.;DeWoody, J. A.
通讯作者：
DeWoody, J. A.
影响因子：
4.8
作者：
HURLBERT, SH
通讯作者：
HURLBERT, SH
影响因子：
4.4
作者：
Krasnov A;Koskinen H;Afanasyev S;Mölsä H
通讯作者：
Mölsä H