High throughput direct end sequencing of BAC clones
High throughput direct end sequencing of BAC clones
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DOI：
10.1093/nar/27.6.1539
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发表时间：
1999-03-15
影响因子：
14.9
通讯作者：
Adams, MD
中科院分区：
文献类型：
--
作者：
Kelley, JM;Field, CE;Adams, MD
在细菌人工染色体（BAC）载体中构建的文库已成为高通量基因组测序项目中克隆集的选择，主要是因为它们的高稳定性。 BAC文库已被提议作为用于对大基因组区域进行测序的最小重叠克隆的来源，并且已提议使用BAC末端序列（即邻近插入位点的序列）作为选择用于对大基因组区域进行测序的最小重叠克隆的主要手段。为了使该策略有效，需要开发对深度覆盖 BAC 文库中所有克隆进行 BAC 末端测序的高通量方法。在这里，我们描述了一种低成本、高效的 96 孔 BAC 末端测序程序。这些方法使我们能够从人类和拟南芥文库中生成 BAC 末端序列，平均读长 > 450 个碱基，单次测序平均准确度 > 98%。讨论了BAC末端序列在基因组测序中的应用。
Libraries constructed in bacterial artificial chromosome (BAC) vectors have become the choice for clone sets in high throughput genomic sequencing projects primarily because of their high stability. BAC libraries have been proposed as a source for minimally overlapping clones for sequencing large genomic regions, and the use of BAC end sequences (i.e. sequences adjoining the insert sites) has been proposed as a primary means for selecting minimally overlapping crones for sequencing large genomic regions. For this strategy to be effective, high throughput methods for BAC end sequencing of all the clones in deep coverage BAC libraries needed to be developed. Here we describe a low cost, efficient, 96 well procedure for BAC end sequencing. These methods allow us to generate BAC end sequences from human and Arabidoposis libraries with an average read length of >450 bases and with a single pass sequencing average accuracy of >98%. Application of BAC end sequences in genomic sequencing is discussed.