Unamplified cap analysis of gene expression on a single-molecule sequencer
Unamplified cap analysis of gene expression on a single-molecule sequencer
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DOI：
10.1101/gr.115469.110
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发表时间：
2011-07-01
期刊：
影响因子：
7
通讯作者：
Hayashizaki, Yoshihide
中科院分区：
文献类型：
--
作者：
Kanamori-Katayama, Mutsumi;Itoh, Masayoshi;Hayashizaki, Yoshihide
我们报告了一种简化的CAGE基因表达分析(CAGE)方案的开发，该方案适用于单分子测序仪，避免了第二链合成、连接、消化和PCR。HeliScope CAGE直接对捕获的帽的3‘端第一链cDNA进行测序。与以前版本的CAGE一样，我们比已知的模型更好地定义了转录起始位点(TSS)，识别了新的转录区域和替代启动子，并发现了TSS信号的两大类，尖锐的峰和广泛的区域。然而，使用这个方案，我们观察到在单个TSS位置的更精细的水平上调节的可重现的证据。文库是定量的，超过5个数量级，重复性高(皮尔逊相关系数为0.987)。我们还将标准HeliScope CAGE文库的样本要求减少到5微克，低量版本的样本要求减少到100毫克。当同样的RNA以5微克和100毫微克版本运行时，100毫微克仍然能够检测到使用相同阈值的5微克文库检测到的13,468个基因座中的60%的表达，这使得即使是稀有细胞群体也可以进行比较分析。在三个HeLa和THP-1样本上测试差异基因表达测量的方法，我们发现与Illumina微阵列测量相比，对数倍变化高度相关(0.871)。此外，HeliScope eCAGE还发现了数千个基因座的差异表达，包括那些在阵列上有探针的基因座。最后，虽然大多数标签是5‘相关的，我们也在外显子上观察到低水平的信号，这对定义基因结构是有用的。
We report the development of a simplified cap analysis of gene expression (CAGE) protocol adapted for single-molecule sequencers that avoids second strand synthesis, ligation, digestion, and PCR. HeliScopeCAGE directly sequences the 3' end of cap trapped first-strand cDNAs. As with previous versions of CAGE, we better define transcription start sites (TSS) than known models, identify novel regions of transcription and alternative promoters, and find two major classes of TSS signal, sharp peaks and broad regions. However, using this protocol, we observe reproducible evidence of regulation at the much finer level of individual TSS positions. The libraries are quantitative over 5 orders of magnitude and highly reproducible (Pearson's correlation coefficient of 0.987). We have also scaled down the sample requirement to 5 mu g of total RNA for a standard HeliScopeCAGE library and 100 ng for a low-quantity version. When the same RNA was run as 5-mu g and 100-ng versions, the 100 ng was still able to detect expression for similar to 60% of the 13,468 loci detected by a 5-mu g library using the same threshold, allowing comparative analysis of even rare cell populations. Testing the protocol for differential gene expression measurements on triplicate HeLa and THP-1 samples, we find that the log fold change compared to Illumina microarray measurements is highly correlated (0.871). In addition, HeliScopeCAGE finds differential expression for thousands more loci including those with probes on the array. Finally, although the majority of tags are 5' associated, we also observe a low level of signal on exons that is useful for defining gene structures.