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首页> 美国卫生研究院文献>Nucleic Acids Research >General method of rapid Smith/Birnstiel mapping adds for gap closure in shotgun microbial genome sequencing projects: application to Pseudomonas putida KT2440
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General method of rapid Smith/Birnstiel mapping adds for gap closure in shotgun microbial genome sequencing projects: application to Pseudomonas putida KT2440

机译：Smith / Birnstiel快速作图的通用方法增加了shot弹枪微生物基因组测序项目中的缺口封闭：在恶臭假单胞菌KT2440中的应用

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摘要

A physical mapping strategy has been developed to verify and accelerate the assembly and gap closure phase of a microbial genome shotgun-sequencing project. The protocol was worked out during the ongoing Pseudomonas putida KT2440 genome project. A macro-restriction map was constructed by linking probe hybridisation of SwaI- or I-CeuI-restricted chromosomes to serve as a backbone for the quick quality control of sequence and contig assemblies. The library of PCR-generated SwaI linking probes was derived from the sequence assembly after 3- and 6-fold genome coverage. In order to support gap closure in regions with ambiguous assemblies such as the repetitive sequence of the seven ribosomal operons, high-resolution Smith/Birnstiel maps were generated by Southern hybridisation of pulsed-field gel electrophoresis-separated rare-cutter complete/frequent-cutter partial digestions with rare-cutter fragment end probes. Overall 1.5 Mb of the 6.1 Mb P.putida KT2440 genome has been subjected to high-resolution physical mapping in order to align assemblies generated from shotgun sequencing.
机译：已经开发了一种物理作图策略，以验证和加速微生物基因组shot弹枪测序项目的组装和缺口封闭阶段。该协议是在正在进行的恶臭假单胞菌KT2440基因组计划中制定的。通过将SwaI或I-CeuI限制性染色体的探针杂交连接起来，构建了一个宏限制性图谱，可作为对序列和重叠群装配进行快速质量控制的骨架。 PCR生成的SwaI连接探针库来自3倍和6倍的基因组覆盖率后的序列装配。为了支持具有歧义装配的区域（例如七个核糖体操纵子的重复序列）中的间隙闭合，通过脉冲场凝胶电泳分离的稀有切割机完整/频繁切割机的Southern杂交生成了高分辨率的Smith / Birnstiel图。使用稀有切割片段末端探针进行部分消化。 6.1 Mb恶臭假单胞菌KT2440基因组中的总1.5 Mb已进行了高分辨率物理作图，以比对由散弹枪测序产生的装配。

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