Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.
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.
Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Michael
0000-0003-1303-5598
M
Baym
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Lev
L
Shaket
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Isao A
IA
Anzai
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Oluwakemi
O
Adesina
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Buz
0000-0003-2218-6152
B
Barstow
D000465:Algorithms; D004251:DNA Transposable Elements / Q000235:genetics*; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D016254:Mutagenesis, Insertional; D009154:Mutation; D020592:Shewanella / Q000235:genetics*
D004251:DNA Transposable Elements