title: Phenotype sequencing: identifying the genes that cause a phenotype directly from pooled sequencing of independent mutants.
abstract: Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50-100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a "Phenotype Sequencing" approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost $7,200 in reagents alone, our Phenotype Sequencing design yielded the same information value for only $1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only $110-$340.
authors.affiliation: Institute for Genomics and Proteomics, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Harper
authors.forename: Zugen
authors.initials: Z
authors.lastname: Chen
authors.forename: Traci
authors.initials: T
authors.lastname: Toy
authors.forename: Iara M P
authors.initials: IM
authors.lastname: Machado
authors.forename: Stanley F
authors.initials: SF
authors.lastname: Nelson
authors.forename: James C
authors.initials: JC
authors.lastname: Liao
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Lee
mesh_terms: D001483:Base Sequence; D000440:Butanols / Q000494:pharmacology; D004361:Drug Tolerance / Q000235:genetics / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D056726:Genetic Association Studies / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D008954:Models, Biological; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000032:analysis / Q000235:genetics*; D009154:Mutation / Q000502:physiology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D030781:Organisms, Genetically Modified; D010641:Phenotype; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D000440:Butanols; D050505:Mutant Proteins; C040507:isobutyl alcohol