Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons

摘要: ABSTRACT Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the are not amenable throughput necessary identify phenotypes and functions majority genes diverse bacteria. Here, we present method, random bar code transposon-site (RB-TnSeq), which increases mutant fitness profiling by incorporating DNA codes into Tn5 mariner transposons using (BarSeq) assay fitness. RB-TnSeq can be used any transposon, performed once per organism instead sample. Each BarSeq requires only simple PCR, 48 96 samples sequenced on one lane an Illumina HiSeq system. We demonstrate reproducibility biological significance Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, oneidensis. To increased RB-TnSeq, 387 successful genome-wide assays representing 130 different bacterium-carbon source combinations identified 5,196 significant across five In P. inhibens, our data important utilization carbon substrates, including putative d-mannose isomerase that required mannitol catabolism. will enable cost-effective functional annotation bacteria profiling. IMPORTANCE A large challenge microbiology assessment millions uncharacterized genome coupled assign genes. However, current strategies too applied hundreds experimental conditions multiple describe approach, greatly simplifies measurement monitor abundance mutants. over 5,000 tool phenotype data.