Hebei Key Laboratory of Soil Ecology, Key Laboratory for Agricultural Water Resource, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050021, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Genes (Basel). 2019 Dec 20;11(1):7. doi: 10.3390/genes11010007.
Functional (meta) genomics allows the high-throughput identification of functional genes in a premise-free way. However, it is still difficult to perform Sanger sequencing for high GC DNA templates, which hinders the functional genomic exploration of a high GC genomic library. Here, we developed a procedure to resolve this problem by coupling the Sanger and PacBio sequencing strategies. Identification of cadmium (Cd) resistance genes from a small-insert high GC genomic library was performed to test the procedure. The library was generated from a high GC (75.35%) bacterial genome. Nineteen clones that conferred Cd resistance to subject to Sanger sequencing directly. The positive clones were in parallel subject to in vivo amplification in host cells, from which recombinant plasmids were extracted and linearized by selected restriction endonucleases. PacBio sequencing was performed to obtain the full-length sequences. As the identities, partial sequences from Sanger sequencing were aligned to the full-length sequences from PacBio sequencing, which led to the identification of seven unique full-length sequences. The unique sequences were further aligned to the full genome sequence of the source strain. Functional screening showed that the identified positive clones were all able to improve Cd resistance of the host cells. The functional genomic procedure developed here couples the Sanger and PacBio sequencing methods and overcomes the difficulties in PCR approaches for high GC DNA. The procedure can be a promising option for the high-throughput sequencing of functional genomic libraries, and realize a cost-effective and time-efficient identification of the positive clones, particularly for high GC genetic materials.
功能（元）基因组学允许在无前提的情况下高通量鉴定功能基因。然而，对于高 GC DNA 模板，仍然难以进行 Sanger 测序，这阻碍了高 GC 基因组文库的功能基因组探索。在这里，我们开发了一种通过结合 Sanger 和 PacBio 测序策略来解决此问题的程序。从一个小插入高 GC 基因组文库中鉴定镉（Cd）抗性基因来测试该程序。该文库由高 GC（75.35%）细菌基因组产生。直接对 19 个赋予 Cd 抗性的克隆进行 Sanger 测序。阳性克隆并行在宿主细胞中进行体内扩增，从这些细胞中提取重组质粒并通过选定的限制性内切酶线性化。进行 PacBio 测序以获得全长序列。由于身份，来自 Sanger 测序的部分序列与来自 PacBio 测序的全长序列对齐，从而鉴定了七个独特的全长序列。进一步将独特序列与来源菌株的全基因组序列对齐。功能筛选表明，鉴定的阳性克隆均能提高宿主细胞的 Cd 抗性。这里开发的功能基因组程序结合了 Sanger 和 PacBio 测序方法，克服了高 GC DNA 中 PCR 方法的困难。该程序可为功能基因组文库的高通量测序提供一种有前途的选择，并实现阳性克隆的经济高效和高效鉴定，特别是对于高 GC 遗传材料。
