An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones
An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones
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DOI：
10.1093/nar/30.11.2460
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发表时间：
2002-06-01
影响因子：
14.9
通讯作者：
Jones, SJM
中科院分区：
文献类型：
--
作者：
Butterfield, YSN;Marra, MA;Jones, SJM
我们描述了一种高效的高通量方法，用于整个cDNA克隆的准确DNA测序。作为我们参与哺乳动物基因收集全长cDNA测序计划的一部分，该方法自2000年9月以来一直在我们的实验室中使用和改进。适合大规模项目，我们使用该方法从3695个候选全长cDNA中生成了>7 Mb的准确序列。测序是通过将Mu转座子插入cDNA中，然后进行用Mu特异性测序引物引发的测序反应来完成的。转座子插入反应不是用单个cDNA进行的，而是在多达96个克隆的库中进行的。这种合并策略减少了原本需要的转座子插入测序文库的数量，降低了成本并提高了转座子文库构建程序的效率。组装使用转座子特异性测序引物产生的序列以产生全长cDNA序列，根据需要进行序列编辑和其他序列整理活动以解决序列模糊性。尽管对成千上万（22785）个测序的Mu转座子插入事件的分析揭示了Mu插入的弱序列偏好，但我们观察到Mu转座子插入到可能的1024个5 mer候选插入位点中的1015个中。
We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites.