Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada.
Nat Methods. 2010 Oct;7(10):843-7. doi: 10.1038/nmeth.1503. Epub 2010 Sep 12.
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/.
在替代表达分析测序 (ALEXA-seq) 中，我们开发了一种分析大规模并行 RNA 序列数据的方法，以对已知和预测的 mRNA 异构体进行转录本分类，并评估细胞和组织中它们的差异表达和选择性表达。作为原理验证，我们使用该方法比较了氟尿嘧啶耐药和非耐药的人结直肠癌细胞系。我们通过与外显子平铺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 Sanger 测序验证结果。我们观察到氟尿嘧啶耐药细胞中剪接的全局破坏，其特征是新的 mRNA 异构体的表达，这些异构体是通过外显子跳跃、选择性剪接位点使用和内含子保留产生的。替代表达注释数据库、源代码、数据查看器和其他便于分析的资源可在 http://www.alexaplatform.org/alexa_seq/ 获得。
