Deep sequencing can accurately measure the relative abundance of hundreds of mutations in a single bulk competition experiment, which can give a direct readout of the fitness of each mutant. Here we describe a protocol that we previously developed and optimized to measure the fitness effects of all possible individual codon substitutions for 10-aa regions of essential genes in yeast. Starting with a conditional strain (i.e., a temperature-sensitive strain), we describe how to efficiently generate plasmid libraries of point mutants that can then be transformed to generate libraries of yeast. The yeast libraries are competed under conditions that select for mutant function. Deep-sequencing analyses are used to determine the relative fitness of all mutants. This approach is faster and cheaper per mutant compared with analyzing individually isolated mutants. The protocol can be performed in ∼4 weeks and many 10-aa regions can be analyzed in parallel.
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| Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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| Site | Modification | Modifier | Source | Reference |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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| Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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| Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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| Evidence ID | Analyze ID | File | Description |
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