Sequencing the junctions of the kanamycin resistance cassette within the Keio library of gene-deletion mutants

The Keio library of single-gene deletion mutants constructed in Escherichia coli K-12 strain BW25113 is one of the most well-characterised, ordered gene-deletion libraries (Baba et al., 2006). During construction of the Keio library, each annotated gene of E. coli BW25113 was targeted for replacement with a kanamycin resistance cassette. Here, we validated the accuracy of the Keio mutants by pooling the mutants and sequencing out from the resistance cassette into genomic DNA, using a multi-parallel sequencing approach. The raw data were trimmed of barcodes and kanamycin resistance cassette sequences, and mapped to the BW25113 reference genome (accession number CP009273.1) to identify the insertion loci of the kanamycin resistance cassette for every Keio mutant.