LI-Detector: a Method for Curating Ordered Gene-Replacement Libraries.
LI-Detector: a Method for Curating Ordered Gene-Replacement Libraries.
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DOI：
10.1128/spectrum.00833-22
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发表时间：
2022-08-31
影响因子：
3.7
通讯作者：
Henderson, Ian R.
中科院分区：
文献类型：
--
作者：
Goodall, Emily C. A.;Morris, Faye C.;McKeand, Samantha A.;Sullivan, Rudi;Warner, Isabel A.;Sheehan, Emma;Boelter, Gabriela;Icke, Christopher;Cunningham, Adam F.;Cole, Jeffrey A.;Banzhaf, Manuel;Bryant, Jack A.;Henderson, Ian R.
关键词：
近年来，基因组序列信息的可获得性已迅速增长，导致大量未表征基因的鉴定。为了解决功能注释中的这一差距，已经设计了许多高通量筛选来发现新的基因功能。基因置换文库就是这样一种工具，它可以以高通量的方式筛选，以连接基因型和表型，是关键的社区资源。然而，对于归因于特定基因的表型，需要对基因型有信心。构建大型库可能很费力，偶尔会出现错误。在这里，我们提出了一种快速，准确的方法，用于验证任何有序的文库，其中基因已被替换或破坏的均匀线性插入（LI）。我们应用我们的方法（LI-检测器），以著名的庆应义塾图书馆的大肠杆菌基因缺失突变体。我们的方法从总共3,728个确认的分离株中鉴定出3,718个构建的突变体，鉴定正确卡那霉素盒位置的成功率为99.7%。该数据集为Keio突变体的纯度提供了基准，并为定位任何线性插入的位置提供了筛选方法，例如任何有序文库中的抗生素抗性盒。重要性有序基因替代文库的构建需要大量的时间和资源投入，以创建有价值的社区资源。在构建过程中，技术错误可能导致有限数量的错误突变体。这些突变体可能会混淆后续实验的结果。在这里，使用非凡的E。coliKeio敲除文库中，我们描述了一种快速验证每个突变体构建的方法。
In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterized genes. To address this gap in functional annotation, many high-throughput screens have been devised to uncover novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered library where a gene has been replaced or disrupted by a uniform linear insertion (LI). We applied our method (LI-detector) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates, with a success rate of 99.7% for identifying the correct kanamycin cassette position. This data set provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of any linear insertion, such as an antibiotic resistance cassette in any ordered library. IMPORTANCE The construction of ordered gene replacement libraries requires significant investment of time and resources to create a valuable community resource. During construction, technical errors may result in a limited number of incorrect mutants being made. Such mutants may confound the output of subsequent experiments. Here, using the remarkable E. coli Keio knockout library, we describe a method to rapidly validate the construction of every mutant.
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DOI：
10.1128/genomea.01038-14
发表时间：
2014-10-16
期刊：
Genome announcements
影响因子：
--
作者：
Grenier F;Matteau D;Baby V;Rodrigue S
通讯作者：
Rodrigue S
影响因子：
4.5
作者：
Goodall ECA;Isom GL;Rooke JL;Pullela K;Icke C;Yang Z;Boelter G;Jones A;Warner I;Da Costa R;Zhang B;Rae J;Tan WB;Winkle M;Delhaye A;Heinz E;Collet JF;Cunningham AF;Blaskovich MA;Parton RG;Cole JA;Banzhaf M;Chng SS;Vollmer W;Bryant JA;Henderson IR
通讯作者：
Henderson IR
影响因子：
5.4
作者：
Gray AN;Koo BM;Shiver AL;Peters JM;Osadnik H;Gross CA
通讯作者：
Gross CA
影响因子：
7
作者：
Langridge, Gemma C.;Phan, Minh-Duy;Turner, A. Keith
通讯作者：
Turner, A. Keith
DOI：
10.1073/pnas.0803281105
发表时间：
2008-06-24
影响因子：
11.1
作者：
Cameron, D. Ewen;Urbach, Jonathan M.;Mekalanos, John J.
通讯作者：
Mekalanos, John J.