Molecular, Cellular and Developmental Biology Department, KBT918, Yale University, New Haven, Connecticut 06511, USA.
Genome Biol. 2008 Jan 3;9(1):R3. doi: 10.1186/gb-2008-9-1-r3.
Recent studies of the mammalian transcriptome have revealed a large number of additional transcribed regions and extraordinary complexity in transcript diversity. However, there is still much uncertainty regarding precisely what portion of the genome is transcribed, the exact structures of these novel transcripts, and the levels of the transcripts produced.
We have interrogated the transcribed loci in 420 selected ENCyclopedia Of DNA Elements (ENCODE) regions using rapid amplification of cDNA ends (RACE) sequencing. We analyzed annotated known gene regions, but primarily we focused on novel transcriptionally active regions (TARs), which were previously identified by high-density oligonucleotide tiling arrays and on random regions that were not believed to be transcribed. We found RACE sequencing to be very sensitive and were able to detect low levels of transcripts in specific cell types that were not detectable by microarrays. We also observed many instances of sense-antisense transcripts; further analysis suggests that many of the antisense transcripts (but not all) may be artifacts generated from the reverse transcription reaction. Our results show that the majority of the novel TARs analyzed (60%) are connected to other novel TARs or known exons. Of previously unannotated random regions, 17% were shown to produce overlapping transcripts. Furthermore, it is estimated that 9% of the novel transcripts encode proteins.
We conclude that RACE sequencing is an efficient, sensitive, and highly accurate method for characterization of the transcriptome of specific cell/tissue types. Using this method, it appears that much of the genome is represented in polyA+ RNA. Moreover, a fraction of the novel RNAs can encode protein and are likely to be functional.
最近对哺乳动物转录组的研究揭示了大量额外的转录区域和转录多样性的非凡复杂性。然而，对于确切哪些基因组部分被转录、这些新转录本的确切结构以及产生的转录本的水平，仍然存在很大的不确定性。
我们使用快速扩增 cDNA 末端（RACE）测序技术，对 420 个选定的 ENCyclopedia Of DNA Elements（ENCODE）区域中的转录基因座进行了检测。我们分析了注释的已知基因区域，但主要关注新的转录活性区域（TAR），这些区域先前是通过高密度寡核苷酸平铺阵列和未被认为转录的随机区域来识别的。我们发现 RACE 测序非常敏感，能够检测到在特定细胞类型中微阵列无法检测到的低水平转录物。我们还观察到许多有意义的反义转录物；进一步的分析表明，许多反义转录物（但不是全部）可能是反转录反应产生的假象。我们的结果表明，分析的大多数新 TAR（60%）与其他新 TAR 或已知外显子相连。以前未注释的随机区域中，有 17%显示出产生重叠转录物。此外，估计 9%的新转录物编码蛋白质。
我们得出结论，RACE 测序是一种高效、敏感和高度准确的方法，用于特定细胞/组织类型的转录组特征描述。使用这种方法，似乎大部分基因组都存在于 polyA+RNA 中。此外，一些新的 RNA 可以编码蛋白质，并且可能具有功能。
