Next-generation tag sequencing for cancer gene expression profiling
Next-generation tag sequencing for cancer gene expression profiling
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DOI：
10.1101/gr.094482.109
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发表时间：
2009-10-01
期刊：
影响因子：
7
通讯作者：
Marra, Marco A.
中科院分区：
文献类型：
--
作者：
Morrissy, A. Sorana;Morin, Ryan D.;Marra, Marco A.
我们描述了一种新的方法，Tag-seq，它采用超高通量测序的21个碱基对的cDNA标签的敏感和成本效益的基因表达谱。我们将Tag-seq数据与LongSAGE数据进行了比较，并观察到几类罕见转录本的代表性有所改善，包括转录因子，反义转录本和内含子序列，后者可能代表新的外显子或基因。我们观察到这种罕见转录本的多样性、丰度和动态范围增加，并利用更大的动态表达范围来鉴定癌症和正常文库中替代转录本同种型的表达比率改变。Tag-seq读数的链特异性信息进一步允许我们检测癌症和正常文库之间有义和反义（S-AS）转录物的改变的表达比率。S-AS转录物在已知的癌症基因中富集，而转录物同种型在miRNA靶向位点中富集。我们发现LongSAGE中的转录本丰度比Tag-seq中具有更强的GC偏好，使得在LongSAGE中富含AT的标签比富含GC的标签丰度低。Tag-seq在基因发现方面也表现得更好，识别了LongSAGE检测到的>98%的基因，并分析了以富含AT基因为特征的转录组的一个独特子集，其表达水平低于LongSAGE检测到的水平。总的来说，Tag-seq对罕见的转录物敏感，相对于LongSAGE具有更小的序列组成偏差，并且允许对更大范围的转录物进行差异表达分析，包括编码重要调控分子的转录物。
We describe a new method, Tag-seq, which employs ultra high-throughput sequencing of 21 base pair cDNA tags for sensitive and cost-effective gene expression profiling. We compared Tag-seq data to LongSAGE data and observed improved representation of several classes of rare transcripts, including transcription factors, antisense transcripts, and intronic sequences, the latter possibly representing novel exons or genes. We observed increases in the diversity, abundance, and dynamic range of such rare transcripts and took advantage of the greater dynamic range of expression to identify, in cancers and normal libraries, altered expression ratios of alternative transcript isoforms. The strand-specific information of Tag-seq reads further allowed us to detect altered expression ratios of sense and antisense (S-AS) transcripts between cancer and normal libraries. S-AS transcripts were enriched in known cancer genes, while transcript isoforms were enriched in miRNA targeting sites. We found that transcript abundance had a stronger GC-bias in LongSAGE than Tag-seq, such that AT-rich tags were less abundant than GC-rich tags in LongSAGE. Tag-seq also performed better in gene discovery, identifying >98% of genes detected by LongSAGE and profiling a distinct subset of the transcriptome characterized by AT-rich genes, which was expressed at levels below those detectable by LongSAGE. Overall, Tag-seq is sensitive to rare transcripts, has less sequence composition bias relative to LongSAGE, and allows differential expression analysis for a greater range of transcripts, including transcripts encoding important regulatory molecules.