Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes.
Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes.
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DOI：
10.1093/nar/gkl444
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发表时间：
2006-07-13
影响因子：
14.9
通讯作者：
Ruan Y
中科院分区：
文献类型：
--
作者：
Ng P;Tan JJ;Ooi HS;Lee YL;Chiu KP;Fullwood MJ;Srinivasan KG;Perbost C;Du L;Sung WK;Wei CL;Ruan Y
配对末端双标签（PET）技术已被证明是有效和准确的大规模转录组和基因组分析。然而，与其他基于DNA标签的测序策略一样，它受到当前桑格技术效率的限制。最近开发的使用皮升规模反应的多重测序方法（454-sequencing™）已经在效率上取得了显著的进步，但是遭受短读取长度和缺乏配对末端信息。为了进一步提高PET分析的效率，同时克服新测序方法的缺点，我们将多重测序与配对末端双标签（MS-PET）结合起来，使用改良的PET程序同时对20万至30万个二聚PET（diPET）模板进行测序，在单个4小时机器运行中输出近50万个PET序列。我们通过分析人乳腺癌细胞的转录组，并通过绘制人结直肠癌细胞基因组中的p53结合位点，证明了MS-PET的实用性和鲁棒性。这种组合的测序策略实现了比当前PET分析标准大约100倍的效率提高，并且还使得短读长多重测序程序能够从大DNA片段获得配对末端信息。
The paired-end ditagging (PET) technique has been shown to be efficient and accurate for large-scale transcriptome and genome analysis. However, as with other DNA tag-based sequencing strategies, it is constrained by the current efficiency of Sanger technology. A recently developed multiplex sequencing method (454-sequencing™) using picolitre-scale reactions has achieved a remarkable advance in efficiency, but suffers from short-read lengths, and a lack of paired-end information. To further enhance the efficiency of PET analysis and at the same time overcome the drawbacks of the new sequencing method, we coupled multiplex sequencing with paired-end ditagging (MS-PET) using modified PET procedures to simultaneously sequence 200 000 to 300 000 dimerized PET (diPET) templates, with an output of nearly half-a-million PET sequences in a single 4 h machine run. We demonstrate the utility and robustness of MS-PET by analyzing the transcriptome of human breast carcinoma cells, and by mapping p53 binding sites in the genome of human colorectal carcinoma cells. This combined sequencing strategy achieved an approximate 100-fold efficiency increase over the current standard for PET analysis, and furthermore enables the short-read-length multiplex sequencing procedure to acquire paired-end information from large DNA fragments.
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