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Nat Protoc. 2011 Nov 17;6(12):1969-80. doi: 10.1038/nprot.2011.417.

Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries.

Author information

1
Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri, USA. andrew.goodman@yale.edu

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.

PMID:
22094732
PMCID:
PMC3310428
DOI:
10.1038/nprot.2011.417
[Indexed for MEDLINE]
Free PMC Article

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