title: Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries.
abstract: Insertion sequencing (INSeq) is a method for determining the insertion site and relative abundance of large numbers of transposon mutants in a mixed population of isogenic mutants of a sequenced microbial species. INSeq is based on a modified mariner transposon containing MmeI sites at its ends, allowing cleavage at chromosomal sites 16-17 bp from the inserted transposon. Genomic regions adjacent to the transposons are amplified by linear PCR with a biotinylated primer. Products are bound to magnetic beads, digested with MmeI and barcoded with sample-specific linkers appended to each restriction fragment. After limited PCR amplification, fragments are sequenced using a high-throughput instrument. The sequence of each read can be used to map the location of a transposon in the genome. Read count measures the relative abundance of that mutant in the population. Solid-phase library preparation makes this protocol rapid (18 h), easy to scale up, amenable to automation and useful for a variety of samples. A protocol for characterizing libraries of transposon mutant strains clonally arrayed in a multiwell format is provided.
authors.affiliation: Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri, USA. andrew.goodman@yale.edu
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Goodman
authors.forename: Meng
authors.initials: M
authors.lastname: Wu
authors.forename: Jeffrey I
authors.initials: JI
authors.lastname: Gordon
mesh_terms: D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry; D015723:Gene Library; D016254:Mutagenesis, Insertional; D009154:Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial
references.citation: Cell Host Microbe. 2009 Sep 17;6(3):279-89
references.pmid: 19748469
references.citation: Genome Res. 2009 Jul;19(7):1243-53
references.pmid: 19447965
references.citation: Genome Biol. 2009;10(3):R25
references.pmid: 19261174
references.citation: Nucleic Acids Res. 2008 Nov;36(20):6558-70
references.pmid: 18931376
references.citation: Bioinformatics. 2005 Apr 1;21(7):880-8
references.pmid: 15539453
references.citation: Curr Protoc Microbiol. 2010 Nov;Chapter 1:Unit1E.3
references.pmid: 21053251
references.citation: Genome Res. 2009 Jul;19(7):1262-9
references.pmid: 19447967
references.citation: Biotechniques. 2004 Feb;36(2):214-6
references.pmid: 14989083
references.citation: Genome Res. 2009 Jul;19(7):1254-61
references.pmid: 19447964
references.citation: Nat Rev Genet. 2006 Dec;7(12):929-39
references.pmid: 17139324
references.citation: Genome Res. 2009 Dec;19(12):2308-16
references.pmid: 19826075
references.citation: mBio. 2011 Jan 18;2(1):e00315-10
references.pmid: 21253457
references.citation: Proc Natl Acad Sci U S A. 2009 Sep 22;106(38):16422-7
references.pmid: 19805314
references.citation: Nat Methods. 2009 Oct;6(10):767-72
references.pmid: 19767758