Unknown

Dataset Information

0

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.

SUBMITTER: Goodman AL 

PROVIDER: S-EPMC3310428 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

altmetric image

Publications

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

Goodman Andrew L AL   Wu Meng M   Gordon Jeffrey I JI  

Nature protocols 20111117 12


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 mag  ...[more]

Similar Datasets

| S-EPMC8894413 | biostudies-literature
| S-EPMC4222735 | biostudies-literature
| S-EPMC5626973 | biostudies-literature
| S-EPMC3597147 | biostudies-literature
| S-EPMC4010991 | biostudies-literature
| S-EPMC4896371 | biostudies-literature
| S-EPMC8256429 | biostudies-literature
| S-EPMC4418687 | biostudies-literature
| S-EPMC5100919 | biostudies-literature
| S-EPMC7007522 | biostudies-literature