Alternative expression analysis by RNA sequencing
Alternative expression analysis by RNA sequencing
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DOI：
10.1038/nmeth.1503
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发表时间：
2010-10-01
期刊：
影响因子：
48
通讯作者：
Marra, Marco A.
中科院分区：
文献类型：
--
作者：
Griffith, Malachi;Griffith, Obi L.;Marra, Marco A.
在测序替代表达分析（ALEXA-seq）中，我们开发了一种方法来分析大规模平行RNA序列数据，以分类转录本并评估细胞和组织中已知和预测的mRNA亚型的差异和替代表达。作为原理的证明，我们使用该方法来比较氟尿嘧啶耐药和非耐药的人结直肠癌细胞系。我们通过与外显子拼接和剪接微阵列的比较来评估该方法的灵敏度和特异性，并通过逆转录PCR、定量PCR和桑格测序来验证结果。我们观察到全球中断的剪接耐药细胞的特点是新的mRNA亚型的表达，导致外显子跳跃，选择性剪接位点的使用和内含子保留。可在http://www.alexaplatform.org/alexa_seq/获得替代表达式注释数据库、源代码、数据查看器和其他资源以促进分析。
In alternative expression analysis by sequencing (ALEXA-seq), we developed a method to analyze massively parallel RNA sequence data to catalog transcripts and assess differential and alternative expression of known and predicted mRNA isoforms in cells and tissues. As proof of principle, we used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines. We assessed the sensitivity and specificity of the approach by comparison to exon tiling and splicing microarrays and validated the results with reverse transcription-PCR, quantitative PCR and Sanger sequencing. We observed global disruption of splicing in fluorouracil-resistant cells characterized by expression of new mRNA isoforms resulting from exon skipping, alternative splice site usage and intron retention. Alternative expression annotation databases, source code, a data viewer and other resources to facilitate analysis are available at http://www.alexaplatform.org/alexa_seq/.