A method for authenticating the fidelity of knockout collections.

Gene knockout strain collections are important tools for discovery in microbiology. The only available genome-wide deletion collection for a human pathogenic fungus, , is utilized widely for genetic studies. We uncovered mix-ups in the assembly of the commercially available deletion collection of ~6,000 unique strains acquired by our lab. While pursuing the characterization of a gene-of-interest, the corresponding deletion strain from the KO collection displayed several interesting phenotypes associated with virulence. However, RNAseq analysis identified transcripts for the putative knockout gene, and the absence of transcripts for a different knockout strain found in the same plate position in an earlier partial knockout collection, raising the possibility that plates from one collection were substituted for the other, indicating a misidentified knockout strain. This was supported by determining the size of the nourseothricin (NAT)-resistance cassette used to generate the two separate knockout libraries. Here we report that our KN99ɑ collection is comprised of mixed plates from two independent libraries and present a simple authentication method that other investigators can use to distinguish the identities of these KO collections.