Rapid whole-genome mutational profiling using next-generation sequencing technologies
Rapid whole-genome mutational profiling using next-generation sequencing technologies
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DOI：
10.1101/gr.077776.108
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发表时间：
2008-10-01
期刊：
影响因子：
7
通讯作者：
Richardson, Paul M.
中科院分区：
文献类型：
--
作者：
Smith, Douglas R.;Quinlan, Aaron R.;Richardson, Paul M.
正向遗传突变研究、适应性进化和表型筛选是创造具有所需性状的新变异生物的有力工具。然而，该过程中产生的突变无法使用传统遗传工具轻易识别。我们表明，新的高通量，大规模并行测序技术可以完全和准确地表征相对于先前测序的亲本（参考）菌株的突变体基因组。我们研究了树干毕赤酵母的突变菌株，该酵母能够将木糖转化为乙醇。这种异常高效的突变菌株是在七年的时间里通过反复的化学诱变、菌株选择、转化和遗传操作开发出来的。我们在三个不同的测序平台上对该菌株进行了重新测序。令人惊讶的是，我们在开放阅读框架中发现了不到12个突变。所有三种测序技术都能够鉴定每个单核苷酸突变，给出至少10-15倍的标称序列覆盖率。我们的研究结果表明，使用新的测序技术，在全基因组水平上检测进化和工程生物中的突变是快速和具有成本效益的。在具有改变的表型的菌株中鉴定特定突变将增加对特定基因功能的了解，并指导进一步的代谢工程努力。
Forward genetic mutational studies, adaptive evolution, and phenotypic screening are powerful tools for creating new variant organisms with desirable traits. However, mutations generated in the process cannot be easily identified with traditional genetic tools. We show that new high-throughput, massively parallel sequencing technologies can completely and accurately characterize a mutant genome relative to a previously sequenced parental (reference) strain. We studied a mutant strain of Pichia stipitis, a yeast capable of converting xylose to ethanol. This unusually efficient mutant strain was developed through repeated rounds of chemical mutagenesis, strain selection, transformation, and genetic manipulation over a period of seven years. We resequenced this strain on three different sequencing platforms. Surprisingly, we found fewer than a dozen mutations in open reading frames. All three sequencing technologies were able to identify each single nucleotide mutation given at least 10-15-fold nominal sequence coverage. Our results show that detecting mutations in evolved and engineered organisms is rapid and cost-effective at the whole-genome level using new sequencing technologies. Identification of specific mutations in strains with altered phenotypes will add insight into specific gene functions and guide further metabolic engineering efforts.