Title | Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku. |
Publication Type | Journal Article |
Year of Publication | 2016 |
Authors | Baym, M, Shaket, L, Anzai, IA, Adesina, O, Barstow, B |
Journal | Nat Commun |
Volume | 7 |
Pagination | 13270 |
Date Published | 2016 11 10 |
ISSN | 2041-1723 |
Keywords | Algorithms, DNA Transposable Elements, Gene Library, Genes, Bacterial, Genome, Bacterial, High-Throughput Nucleotide Sequencing, Mutagenesis, Insertional, Mutation, Shewanella |
Abstract | <p>Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.</p> |
DOI | 10.1038/ncomms13270 |
Alternate Journal | Nat Commun |
PubMed ID | 27830751 |
PubMed Central ID | PMC5109470 |