Direct RNA sequencing
Direct RNA sequencing
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DOI：
10.1038/nature08390
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发表时间：
2009-10-08
期刊：
影响因子：
64.8
通讯作者：
Milos, Patrice M.
中科院分区：
文献类型：
--
作者：
Ozsolak, Fatih;Platt, Adam R.;Milos, Patrice M.
我们对人类生物学和疾病的理解最终取决于对基因组及其功能的全面理解。微阵列和测序技术在转录组学中的近期应用，已将对转录组的简单看法转变为对全基因组转录的更复杂看法，其中很大一部分转录本来自基因组的未注释区域（1 - 7），并突显了我们对转录动态状态的有限了解。大部分这类广泛的知识是间接获得的，因为当前的转录组分析方法通常要求在测量前将RNA转化为互补DNA（cDNA），尽管cDNA合成步骤会引入多种偏差和人为假象，这些都会干扰转录本的正确定性和定量（8 - 18）。此外，cDNA合成并不特别适用于分析短的、降解的和/或少量的RNA样本。在此我们报道了无需事先将RNA转化为cDNA的直接单分子RNA测序。我们应用这项技术对飞摩尔量的酿酒酵母多聚（A）⁺ RNA进行测序，使用涂有聚（dT）寡核苷酸的表面在RNA的天然多聚（A）尾部捕获RNA，并通过合成启动测序。我们观察到转录本3'端的异质性以及多聚腺苷酸化的小核仁RNA。这项研究为高通量、低成本的直接RNA测序提供了一条途径，并朝着全面且无偏差地理解转录组这一最终目标迈进。
Our understanding of human biology and disease is ultimately dependent on a complete understanding of the genome and its functions. The recent application of microarray and sequencing technologies to transcriptomics has changed the simplistic view of transcriptomes to a more complicated view of genome-wide transcription where a large fraction of transcripts emanates from unannotated parts of genomes(1-7), and underlined our limited knowledge of the dynamic state of transcription. Most of this broad body of knowledge was obtained indirectly because current transcriptome analysis methods typically require RNA to be converted to complementary DNA ( cDNA) before measurements, even though the cDNA synthesis step introduces multiple biases and artefacts that interfere with both the proper characterization and quantification of transcripts(8-18). Furthermore, cDNA synthesis is not particularly suitable for the analysis of short, degraded and/or small quantity RNA samples. Here we report direct single molecule RNA sequencing without prior conversion of RNA to cDNA. We applied this technology to sequence femtomole quantities of poly(A)(+) Saccharomyces cerevisiae RNA using a surface coated with poly(dT) oligonucleotides to capture the RNAs at their natural poly(A) tails and initiate sequencing by synthesis. We observed transcript 3' end heterogeneity and polyadenylated small nucleolar RNAs. This study provides a path to high-throughput and low-cost direct RNA sequencing and achieving the ultimate goal of a comprehensive and bias-free understanding of transcriptomes.