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Genetic Screen: Scaling up Mutant Generation Processes

Dec 22, 2019 | Insights

High-throughput genetic screens can produce large data sets that a give system-level understanding of a regulatory network. They require standardization and optimization of data generation and data analysis workflows.

High-Throughput Genetic Screen

Problem

Goal: The client was interested in better understanding a regulatory network. Since no large-scale genetic screen had been published, the client was heavily relying on small data sets published by various groups using multiple yeast strains and methods. It was impossible integrate various data sources to gain systems-level understanding of the network. In order to develop a system-level understanding of a complex regulatory network they run a high-throughput genetic screen to characterize the genetic interactions between the 36 genes involved in this pathway. Genetic interactions are non-linear effects resulting from the combination of multiple mutations.

Problem: While the client has expertise in yeast genetics, they were unable to design workflows suitable to generate and test the library of 1200 mutants needed to gain complete understanding of the regulatory network.

They needed help to scale up the genetic screen process.

Solution

GenoFAB worked with the client to:

  • Design
    • 180 selection cassettes by combining 36 homologous recombination sequences and 5 selection cassettes for each of the 36 genes.
    • 360 primers to generate 180 DNA fragments by amplifying selection cassettes from existing plasmids
    • Sets of primers to verify parent lines by PCR.
  • Optimize and standardize
    • Yeast transformation and yeast mating workflows for stability and reproducibility prior to scale up to minimize the cost of failed experiments.
    • Microtiter plate designs to facilitate automation allowing the generation of more than 5000 crosses.
    • Workflow to allow technicians with little prior molecular biology experience to complete the work.
    • Workflow to maximize data quality while avoiding unmanageable workloads.
    • Testing of more than 5000 mutants by measuring colony size at 6 different time points on 6 different media.
    • Design plate structure to ensure that controls necessary for the analysis were on all plates.
  • Automate
    • Generation of barcode labels and uploading of data to Excel spreadsheets.
    • Statistical analysis of colony size on the plate, including normalization based on the distance of individual colonies from the plate edge.
    • Analysis of growth rate of colonies of individual mutants compared to the growth rate of the control strain, filtering to eliminate outlier colonies resulting from pinning errors.
    • Derivation of estimates of fitness from growth rates and estimates of genetic interactions from fitness estimates.
  • Manage
    • Data by creating a data model to capture all project samples in GenoFAB’s LIMS.
    • Electrophoresis data were uploaded in custom database and automatically analyzed.
    • Data by designing a custom database to import data produced by the plate imager and connect plate images with the samples recorded in the LIMS.
    • Project by developing a custom dashboard pulling data out of the LIMS to keep track of the mutant development progress.
    • Results by developing a solution to identify parent strains that did not transform successfully in order to re-run them.

Results

This genetic screen uncovered major reproducibility issues with the qualitative test that was the commonly accepted standard in the field. Through the help and guidance of GenoFAB, the client was able to produce quantitative datasets that are more reproducible than the standard qualitative data. They have produced the most comprehensive set of data available to their community of interest.  Analysis of this new kind of data is ongoing but it is expected to become the new standard in the field.

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