Kelley J M, Field C E, Craven M B, Bocskai D, Kim U J, Rounsley S D, Adams M D
The Institute for Genomic Research, Rockville, MD 20850, USA and Division of Biology,California Institute of Technology, Pasadena, CA 91125, USA.
Nucleic Acids Res. 1999 Mar 15;27(6):1539-46. doi: 10.1093/nar/27.6.1539.
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 over-lapping 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 clones 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 sequen-cing is discussed.
构建在细菌人工染色体（BAC）载体中的文库已成为高通量基因组测序项目中克隆集的首选，主要是因为它们具有高度稳定性。BAC文库已被提议作为用于对大型基因组区域进行测序的最小重叠克隆的来源，并且使用BAC末端序列（即邻接插入位点的序列）已被提议作为选择用于对大型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，需要开发用于对深度覆盖BAC文库中所有克隆进行BAC末端测序的高通量方法。在这里，我们描述了一种用于BAC末端测序的低成本、高效的96孔方法。这些方法使我们能够从人和拟南芥文库中生成平均读长>450个碱基且单次测序平均准确率>98%的BAC末端序列。文中还讨论了BAC末端序列在基因组测序中的应用。
