Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants.
By G. Langridge, M. Phan, D. Turner, Timothy T. Perkins, Leopold Parts, J. Haase, I. Charles, D. Maskell, S. Peters, Gordon Dougan and 3 more. Genome Research, 2009
650 citations recorded by the source corpus.
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.
Subfields: Ecology, Endocrinology, Food Science
View Paper: https://www.semanticscholar.org/paper/8f772d17329d9160c082ae172a72d99b0c9c6b0d
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In Ecology
Citations in the corpus
- A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers (2012, background): “Illumina sequencing has matured to the point where a great many applications [15-24] have been developed on the platform.”
- High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism (2011, methodology): “others have used similar deep sequencing methods to map insertion sites in other organisms [28,29,30,31,32] our new analytical tools allowed the statistically rigorous prediction of the”
- DEG 10, an update of the database of essential genes that includes both protein-coding genes and noncoding genomic elements (2013, background): “In 1999, Venter and coworkers first performed Sanger sequencing to determine transposon insertion sites (12), and later, various versions of combining transposon mutagenesis and next-generation sequencing were developed, such as TraDIS (13), INSeq *To whom correspondence should be addressed.”
- Lag Phase Is a Distinct Growth Phase That Prepares Bacteria for Exponential Growth and Involves Transient Metal Accumulation (2011, methodology): “To assess the pattern of expression of key physiological processes during lag phase, a published list of essential Salmonella Typhi genes previously identified by the TraDIS global mutagenesis technique was used (64).”