据一篇发表于Genome Research的研究报告,Sanger研究所的研究人员开发出一种高通量的基因测序方法——转座子直接插入位点测序法(Transposon Directed Insertion site Sequencing,TraDIS),该方法只需通过一次试验就能完成对伤寒沙门氏菌(Salmonella Typhi)基因组中所有基因的测序工作。

       该课题组对S.Typhi基因组的分析表明,所有基因中有256个基因是该病原菌生存必需的基因,其余4162个基因不是生存所必需的基因。了解哪些是病原生存必需基因有助于研究人员以这些基因为靶标开发出新的治疗方法。

       研究人员通过TraDIS法将转座子插入到S.Typhi基因组中,产生了超过1百万个突变体。再将这些突变体细菌进行培养,通过对这些病原菌子代的基因组进行测序,发现整个S.  Typhi基因组中有370,000个转座子插入位点——平均每个基因有超过80个插入位点。

       如果转座子插入到生存必需的基因中,将会引起该基因沉默,而且该突变体细胞将不能生长。通过对整个突变库的DNA测序,课题组能够识别所有未插入转座子的基因。

       通过利用TraDIS法,该课题组一次性的出S.Typhi基因组中99.6%对该病原菌生存必需的基因。

 

生物谷推荐原始出处:

Genome Research October 13, 2009, doi:10.1101/gr.097097.109

Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants

Gemma C. Langridge1,6, Minh-Duy Phan1,6, Daniel J. Turner1,6, Timothy T. Perkins1, Leopold Parts1, Jana Haase2, Ian Charles3, Duncan J. Maskell4, Sarah E. Peters4, Gordon Dougan1, John Wain5, Julian Parkhill1,7 and A. Keith Turner1

1The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom;
2Environmental Research Institute, University College, Cork, Ireland;
3Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom;
4Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom;
5Laboratory of Gastrointestinal Pathogens, Centre for Infections, Health Protection Agency, Colindale, London NW9 5HT, United Kingdom

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 semiquantitative 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.