High-Resolution Analysis of the 5′-End Transcriptome Using a Next Generation DNA Sequencer
High-Resolution Analysis of the 5′-End Transcriptome Using a Next Generation DNA Sequencer
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DOI：
10.1371/journal.pone.0004108
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发表时间：
2009-01-01
期刊：
影响因子：
3.7
通讯作者：
Matsushima, Kouji
中科院分区：
文献类型：
--
作者：
Hashimoto, Shin-ichi;Qu, Wei;Matsushima, Kouji
大规模并行、基于标签的测序系统（例如 SOLiD 系统）有望彻底改变全基因组基因表达的研究，因为可以以简单且经济高效的方式生成大量数据点。我们描述了 SOLiD 系统 5' 端转录组工作流程的开发，并证明了这种基于标签的应用程序相对于全基因组基因表达研究的传统方法在灵敏度和动态范围方面的优势。 5' 端转录组分析用于研究经 DNA 甲基转移酶抑制剂 5-aza-2'-脱氧胞苷 (5Aza) 处理的结肠癌细胞系 HT-29 内的全基因组基因表达。从未经处理和 5Aza 处理的细胞中获得了超过 2000 万个 25 碱基 5' 末端标签，并与人类基因组内的序列进行匹配。 73% 的映射独特标签与 RefSeq cDNA 序列相关，对应于该单一细胞类型中大约 14,000 个不同的蛋白质编码基因。这些基因的表达水平范围为每个细胞 0.02 至 4,704 个转录本。 SOLiD 平台单次序列运行的灵敏度比通过 Sanger 测序获得的 70,000 个标签分析生成的 5' 端 SAGE 数据观察到的灵敏度高 100-1,000 倍。本研究中提出的高分辨率 5' 末端基因表达谱不仅将为转录机制提供新的见解，而且还应作为更好地理解细胞生物学的基础。
Massively parallel, tag-based sequencing systems, such as the SOLiD system, hold the promise of revolutionizing the study of whole genome gene expression due to the number of data points that can be generated in a simple and cost-effective manner. We describe the development of a 5'-end transcriptome workflow for the SOLiD system and demonstrate the advantages in sensitivity and dynamic range offered by this tag-based application over traditional approaches for the study of whole genome gene expression. 5'-end transcriptome analysis was used to study whole genome gene expression within a colon cancer cell line, HT-29, treated with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5Aza). More than 20 million 25-base 5'-end tags were obtained from untreated and 5Aza-treated cells and matched to sequences within the human genome. Seventy three percent of the mapped unique tags were associated with RefSeq cDNA sequences, corresponding to approximately 14,000 different protein-coding genes in this single cell type. The level of expression of these genes ranged from 0.02 to 4,704 transcripts per cell. The sensitivity of a single sequence run of the SOLiD platform was 100-1,000 fold greater than that observed from 5' end SAGE data generated from the analysis of 70,000 tags obtained by Sanger sequencing. The high-resolution 5' end gene expression profiling presented in this study will not only provide novel insight into the transcriptional machinery but should also serve as a basis for a better understanding of cell biology.