Phenotype Sequencing: Identifying the Genes That Cause a Phenotype Directly from Pooled Sequencing of Independent Mutants
Phenotype Sequencing: Identifying the Genes That Cause a Phenotype Directly from Pooled Sequencing of Independent Mutants
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DOI：
10.1371/journal.pone.0016517
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发表时间：
2011-02-18
期刊：
影响因子：
3.7
通讯作者：
Lee, Christopher J.
中科院分区：
文献类型：
--
作者：
Harper, Marc A.;Chen, Zugen;Lee, Christopher J.
随机诱变和表型筛选为解剖微生物功能提供了一种强有力的方法，但其结果在实验上分析起来很费力。每个突变株可能包含50-100个随机突变，需要大量的功能实验来确定哪一个导致所选的表型。为了解决这一问题，我们提出了一种“表型测序”方法，该方法可以直接从多个独立突变体的测序中识别引起表型的基因。我们开发了一种新的计算分析方法，表明1。即使从数量不多的突变基因组中，也可以高概率地识别出致病基因；2. 通过优化库池（每个库多个菌株）和标签池（每个测序通道多个标记库）的策略，与传统的基因组测序方法相比，成本可以降低许多倍。我们对一组异丁醇生物燃料耐受性增加的大肠杆菌突变体进行了广泛的验证实验。我们进行了一系列的测序实验，从3到32个突变株，在1到3个测序通道上汇集。我们对这些数据（来自32个突变基因组的4099个突变）进行统计分析，成功地鉴定出3个基因（acrB, marC, acrA），这些基因已被独立验证为导致这种实验表型。必须强调的是，我们的方法极大地降低了突变体测序成本。传统的基因组测序实验仅试剂就需要花费7200美元，而我们的表型测序设计只需要1200美元就能获得相同的信息价值。事实上，我们最小的实验可以可靠地识别acrB和marC，成本仅为110- 340美元。
Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50-100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a "Phenotype Sequencing'' approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost $7,200 in reagents alone, our Phenotype Sequencing design yielded the same information value for only $1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only $110-$340.