TraDIS-validate: a method for curating ordered gene-replacement libraries

Goodall, E. C. A.; Morris, F. C.; McKeand, S. A.; Sullivan, R.; Warner, I. A.; Sheehan, E.; Boelter, G.; Icke, C.; Cunningham, A. F.; Cole, J. A.; Banzhaf, M.; Bryant, J. A.; Henderson, I. R.

doi:10.1101/2022.02.16.479736

bioRxiv · 10.1101/2022.02.16.479736

TraDIS-validate: a method for curating ordered gene-replacement libraries

Goodall, E. C. A.·Morris, F. C.·McKeand, S. A.·Sullivan, R.·Warner, I. A.·Sheehan, E.·Boelter, G.·Icke, C.·Cunningham, A. F.·Cole, J. A.·Banzhaf, M.·Bryant, J. A.·Henderson, I. R.

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Abstract

In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterised genes. To address this gap in functional annotation, many high-throughput screens have been devised with the goal of uncovering novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered gene-replacement library. We applied our method (TraDIS-Validate) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates. TraDIS-validate therefore had a success rate of 99.7% for identifying the correct kanamycin cassette position. This dataset provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of an antibiotic resistance cassette in any ordered library.

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Goodall, E. C. A., Morris, F. C., McKeand, S. A., Sullivan, R., Warner, I. A., Sheehan, E., Boelter, G., Icke, C., Cunningham, A. F., Cole, J. A., Banzhaf, M., Bryant, J. A., Henderson, I. R.. 2022-02-16. TraDIS-validate: a method for curating ordered gene-replacement libraries. https://doi.org/10.1101/2022.02.16.479736

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