A Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in Mycobacterium abscessus.
A Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in Mycobacterium abscessus.
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DOI：
10.1128/msystems.00976-20
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发表时间：
2020-10-20
期刊：
影响因子：
6.4
通讯作者：
Barkan D
中科院分区：
文献类型：
--
作者：
Foreman M;Gershoni M;Barkan D
转座子插入测序是一种强大的工具，但许多研究人员对制备用于深度测序的基因组文库的明显技术复杂性和插入位点鉴定所需的复杂计算分析感到沮丧。我们提出的方法使得文库的制备简单而直接，依赖于众所周知的分子生物学技术。此外，从深度测序获得的结果很容易在转座子插入位点鉴定方面进行分析，将文库制备和分析置于微生物学界更多研究人员的范围内，包括那些具有较少计算和生物信息学资源和经验的研究人员。这是证明了最饱和的Tn突变库的分析，迄今为止创建的新兴病原体分枝杆菌。我们提出了一种技术简单，易于执行的方法来产生的基因组文库的Himar-1转座子位点测序（Tn-seq）。除了在技术方面比现有方法更简单之外，它还允许通过在基因组中产生围绕插入TA的更长序列来更稳健和直接地鉴定插入位点。该方法使Tn-seq更加用户友好，并且可供生物信息资源更有限的实验室使用。最后，我们创建了一个饱和的转座子突变体库中的分枝杆菌，并证明了该方法的有用性，在分析基因参与菌落形态，以及在分析整个Tn突变体库，与超过8,000个独特的突变体的鉴定。转座子插入测序是一种强大的工具，但许多研究人员对准备基因组文库进行深度测序的明显技术复杂性和插入位点鉴定所需的复杂计算分析感到沮丧。我们提出的方法使得文库的制备简单而直接，依赖于众所周知的分子生物学技术。此外，从深度测序获得的结果很容易在转座子插入位点鉴定方面进行分析，将文库制备和分析置于微生物学界更多研究人员的范围内，包括那些具有较少计算和生物信息学资源和经验的研究人员。这是证明了最饱和的Tn突变库的分析，迄今为止创建的新兴病原体分枝杆菌。
Transposon insertion sequencing is a powerful tool, but many researchers are discouraged by the apparent technical complexity of preparing the genomic library for deep sequencing and by the complicated computational analysis needed for insertion site identification. Our proposed method makes the preparation of the library easy and straightforward, relying on well-known molecular biology techniques. In addition, the results obtained from the deep sequencing are easily analyzed in terms of transposon insertion site identification, placing library preparation and analysis within the reach of more researchers in the microbiology community, including those with less computational and bioinformatic resources and experience. This is demonstrated by analysis of the most saturated Tn-mutant library created to date in the emerging pathogen Mycobacterium abscessus. We present a technically simple, easy-to-perform method for generating the genomic libraries for Himar-1 transposon site sequencing (Tn-seq). In addition to being simpler than present methods in the technical aspect, it also allows more robust and straightforward identification of the insertion site, by generating a longer sequence surrounding the insertion TA in the genome. The method makes Tn-seq more user-friendly and accessible to laboratories with more-limited bioinformatic resources. Finally, we created a saturated transposon-mutant library in Mycobacterium abscessus and demonstrated the usefulness of the method in analysis of genes involved in colony morphology, as well as in analysis of the whole Tn-mutant library, with identification of over 8,000 unique mutants. IMPORTANCE Transposon insertion sequencing is a powerful tool, but many researchers are discouraged by the apparent technical complexity of preparing the genomic library for deep sequencing and by the complicated computational analysis needed for insertion site identification. Our proposed method makes the preparation of the library easy and straightforward, relying on well-known molecular biology techniques. In addition, the results obtained from the deep sequencing are easily analyzed in terms of transposon insertion site identification, placing library preparation and analysis within the reach of more researchers in the microbiology community, including those with less computational and bioinformatic resources and experience. This is demonstrated by analysis of the most saturated Tn-mutant library created to date in the emerging pathogen Mycobacterium abscessus.