Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons
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 existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data tomore »
- Lawrence Berkeley National Laboratory, Berkeley, CA (United States). Physical Biosciences Division.
- Lawrence Berkeley National Laboratory, Walnut Creek, CA (United States). Joint Genome Institute.
- Lawrence Berkeley National Laboratory, Berkeley, CA (United States). Life Sciences Division.
- Lawrence Berkeley National Laboratory, Berkeley, CA (United States). Physical Biosciences Division; Univ. of California, Berkeley, CA (United States)
- Publication Date:
- OSTI Identifier:
- Grant/Contract Number:
- Accepted Manuscript
- Journal Name:
- mBio (Online)
- Additional Journal Information:
- Journal Name: mBio (Online); Journal Volume: 6; Journal Issue: 3; Journal ID: ISSN 2150-7511
- American Society for Microbiology
- Research Org:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Org:
- USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
- Country of Publication:
- United States
- 59 BASIC BIOLOGICAL SCIENCES