UCLA Phenotype Sequencing
Phenotype sequencing: identifying the genes that cause a phenotype directly from pooled sequencing of independent mutants
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 identi?ed directly from sequencing of multiple independent mutants.  We developed a new computational analysis method showing that 1. causal genes can be identi?ed 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 identi?ed 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.