Mutation detection with next-generation resequencing through a mediator genome.
Mutation detection with next-generation resequencing through a mediator genome.
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DOI：
10.1371/journal.pone.0015628
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发表时间：
2010-12-31
期刊：
影响因子：
3.7
通讯作者：
Sorek R
中科院分区：
文献类型：
--
作者：
Wurtzel O;Dori-Bachash M;Pietrokovski S;Jurkevitch E;Sorek R
下一代测序（NGS）的可负担性正在改变细菌突变分析领域。表型改变的遗传基础可以通过对突变体的整个基因组进行测序并将其与野生型（WT）基因组进行比较来直接鉴定，从而鉴定获得性突变。这种方法的主要限制是需要WT生物体的先验测序的参考基因组，因为大多数当前NGS方法的短读段通常禁止从头基因组组装。为了克服这一限制，我们提出了一个总体框架，利用相对生物体的基因组作为比较WT和突变细菌的介质。在该框架下，突变体和WT基因组都用NGS测序，并且短测序读段被映射到介体基因组。突变体和WT中重现的介体之间的变化被忽略，从而精确定位突变体和WT之间的差异。为了验证这种方法，我们测序了噬菌蛭弧菌109 J，一个强制性的细菌捕食者，和它的猎物独立的突变体的基因组，并比较了介体物种噬菌蛭弧菌HD100。尽管突变体和介体序列在超过28，000个核苷酸位置上存在差异，但我们的方法能够精确定位单个致病突变。在另外53个突变体中的实验验证进一步确立了所涉及的基因。我们的方法扩展了基于NGS的突变分析的适用性，超出了可用参考基因组的范围。
The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WT and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.
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影响因子：
9.5
作者：
Dalca, Adrian V.;Brudno, Michael
通讯作者：
Brudno, Michael
DOI：
10.1126/science.1182395
发表时间：
2010-01-22
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Harris SR;Feil EJ;Holden MT;Quail MA;Nickerson EK;Chantratita N;Gardete S;Tavares A;Day N;Lindsay JA;Edgeworth JD;de Lencastre H;Parkhill J;Peacock SJ;Bentley SD
通讯作者：
Bentley SD
影响因子：
7
作者：
Li, Heng;Ruan, Jue;Durbin, Richard
通讯作者：
Durbin, Richard
DOI：
10.1073/pnas.0702412104
发表时间：
2007-05-15
影响因子：
11.1
作者：
Mrazek, Jan;Guo, Xiangxue;Shah, Apurva
通讯作者：
Shah, Apurva
影响因子：
7
作者：
Chaisson, Mark J.;Brinza, Dumitru;Pevzner, Pavel A.
通讯作者：
Pevzner, Pavel A.