Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants
Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants
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DOI：
10.1101/gr.097097.109
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发表时间：
2009-12-01
期刊：
影响因子：
7
通讯作者：
Turner, A. Keith
中科院分区：
文献类型：
--
作者：
Langridge, Gemma C.;Phan, Minh-Duy;Turner, A. Keith
如果我们要充分利用目前基因组序列的爆炸性增长，非常高通量的测序技术需要与高通量的功能研究相匹配。我们已经产生了一个非常大的细菌突变体库，由估计110万个转座子突变体组成，我们使用了来自该突变体库的基因组DNA，并使用Illumina核苷酸测序从转座子引发并测序到相邻的靶DNA中。通过这种方法，我们称之为TraDIS（转座子定向插入位点测序），我们已经能够将370，000个独特的转座子插入位点映射到沙门氏菌伤寒血清型染色体上。前所未有的密度和分辨率映射插入位点，平均每13个碱基对一个，使我们能够同时测定基因组中的每个基因的必要性，并产生一个全基因组的候选必需基因列表。此外，半定量性质的测定使我们能够确定基因是有利的，那些是不利的生长在标准实验室条件下。在存在或不存在牛胆汁的情况下生长后，对突变体库进行比较，可以测定每个基因对胆汁耐受性的贡献，胆汁耐受性是任何肠道细菌所需的性状，也是携带S.伤寒在胆囊里。该筛选验证了我们的假设，即我们可以同时测定基因组中的每个基因以鉴定小生境特异性必需基因。
Very high-throughput sequencing technologies need to be matched by high-throughput functional studies if we are to make full use of the current explosion in genome sequences. We have generated a very large bacterial mutant pool, consisting of an estimated 1.1 million transposon mutants and we have used genomic DNA from this mutant pool, and Illumina nucleotide sequencing to prime from the transposon and sequence into the adjacent target DNA. With this method, which we have called TraDIS ( transposon directed insertion-site sequencing), we have been able to map 370,000 unique transposon insertion sites to the Salmonella enterica serovar Typhi chromosome. The unprecedented density and resolution of mapped insertion sites, an average of one every 13 base pairs, has allowed us to assay simultaneously every gene in the genome for essentiality and generate a genome-wide list of candidate essential genes. In addition, the semi-quantitative nature of the assay allowed us to identify genes that are advantageous and those that are disadvantageous for growth under standard laboratory conditions. Comparison of the mutant pool following growth in the presence or absence of ox bile enabled every gene to be assayed for its contribution toward bile tolerance, a trait required of any enteric bacterium and for carriage of S. Typhi in the gall bladder. This screen validated our hypothesis that we can simultaneously assay every gene in the genome to identify niche-specific essential genes.