Gene discovery and annotation using LCM-454 transcriptome sequencing
Gene discovery and annotation using LCM-454 transcriptome sequencing
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DOI：
10.1101/gr.5145806
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发表时间：
2007-01-01
期刊：
影响因子：
7
通讯作者：
Schnable, Patrick S.
中科院分区：
文献类型：
--
作者：
Emrich, Scott J.;Barbazuk, W. Brad;Schnable, Patrick S.
与传统方法相比，454 DNA测序技术实现了显著的吞吐量。454生命科学利用激光捕获显微切割技术(LCM)从玉米(Zea Mays L.)顶端分生组织(SAM)中分离出了超过261,000个EST。这个单一的测序运行注释了25,000个玉米基因组序列，也捕获了400个表达的转录本，这些转录本的同源序列在其他物种中尚未被识别。在这项研究中产生的大约70%的EST在之前的EST项目中没有被捕获到，该项目使用的是一个由手工解剖的顶端组织构建的cDNA文库，该文库对SAM高度浓缩。此外，按照保守的比对标准，454个EST中至少有30%没有与现有的648,000个玉米EST中的任何一个进行比对。这些结果表明，LCM和454技术可能的深度测序相结合，丰富了当前EST样本中没有的SAM转录本。通过LCM-454技术在SAM中检测到27个基因的表达，但在GenBank中没有同源基因，通过RT-PCR验证这些基因的表达。值得注意的是，在富含分生组织的未成熟耳朵中，类似于74%(20/27)的SAM表达的孤儿的转录本没有被检测到。我们的结论是，LCM和454测序技术的结合有助于发现罕见的、可能具有细胞类型特异性的转录本。
454 DNA sequencing technology achieves significant throughput relative to traditional approaches. More than 261,000 ESTs were generated by 454 Life Sciences from cDNA isolated using laser capture microdissection (LCM) from the developmentally important shoot apical meristem (SAM) of maize (Zea mays L.). This single sequencing run annotated > 25,000 maize genomic sequences and also captured similar to 400 expressed transcripts for which homologous sequences have not yet been identified in other species. Approximately 70% of the ESTs generated in this study had not been captured during a previous EST project conducted using a cDNA library constructed from hand-dissected apex tissue that is highly enriched for SAMs. In addition, at least 30% of the 454-ESTs do not align to any of the similar to 648,000 extant maize ESTs using conservative alignment criteria. These results indicate that the combination of LCM and the deep sequencing possible with 454 technology enriches for SAM transcripts not present in current EST collections. RT-PCR was used to validate the expression of 27 genes whose expression had been detected in the SAM via LCM-454 technology, but that lacked orthologs in GenBank. Significantly, transcripts from similar to 74% (20/ 27) of these validated SAM-expressed "orphans" were not detected in meristem-rich immature ears. We conclude that the coupling of LCM and 454 sequencing technologies facilitates the discovery of rare, possibly cell-type-specific transcripts.