Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons.
Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons.
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DOI：
10.1128/mbio.00306-15
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发表时间：
2015-05-12
期刊：
影响因子：
6.4
通讯作者：
Deutschbauer A
中科院分区：
文献类型：
--
作者：
Wetmore KM;Price MN;Waters RJ;Lamson JS;He J;Hoover CA;Blow MJ;Bristow J;Butland G;Arkin AP;Deutschbauer A
转座子突变与下一代测序（TnSeq）是一种强大的方法来注释细菌基因功能，但现有的TnSeq方案需要在测序前对每个样品进行艰苦的准备。因此，现有的方案不能满足鉴定不同细菌中大多数基因的表型和功能所需的吞吐量。在这里，我们提出了一种方法，随机条形码转座子测序（RB-TnSeq），该方法通过将随机DNA条形码结合到Tn5和mariner转座子中，并使用条形码测序（BarSeq）来测定突变适应度，从而提高了突变适应度分析的吞吐量。RB-TnSeq可用于任何转座子，并且TnSeq对每个生物体而不是每个样品进行一次检测。每个BarSeq检测只需要一个简单的PCR，并且在Illumina HiSeq系统的一个通道上可以对48到96个样品进行测序。我们证明了RB-TnSeq在大肠杆菌、抑制费杆菌、假单胞菌、亚马逊希瓦氏菌和同一希瓦氏菌中的重复性和生物学意义。为了证明RB-TnSeq提高了通量，我们进行了387次成功的全基因组突变适应度测定，代表130种不同的细菌-碳源组合，并鉴定出5种细菌中具有显著表型的5196个基因。在P. inhibens中，我们使用突变适应度数据来确定对利用不同碳底物（包括甘露醇分解代谢所需的假定的d-甘露糖异构酶）重要的基因。RB-TnSeq将使用突变适应度分析实现多种细菌的经济高效功能注释。微生物学的一大挑战是对基因组测序鉴定的数百万未表征基因的功能评估。转座子突变与下一代测序（TnSeq）是一种有效的方法来分配基因的表型和功能。然而，目前的TnSeq策略过于费力，无法应用于数百种实验条件下的多种细菌。在这里，我们描述了一种方法，随机条形码转座测序（RB-TnSeq），该方法通过条形码测序（BarSeq）来监测突变体的丰度，极大地简化了基因适应度的测量。我们对5种细菌进行了387次全基因组适应度分析，并确定了5000多个基因的表型。RB-TnSeq可以应用于多种细菌，是使用表型数据注释未表征基因的强大工具。
Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative d-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.