Phenotype Sequencing: Identifying the Genes That Cause a Phenotype Directly from Pooled Sequencing of Independent MutantsReportar como inadecuado




Phenotype Sequencing: Identifying the Genes That Cause a Phenotype Directly from Pooled Sequencing of Independent Mutants - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

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.



Autor: Marc A. Harper, Zugen Chen, Traci Toy, Iara M. P. Machado, Stanley F. Nelson, James C. Liao, Christopher J. Lee

Fuente: http://plos.srce.hr/



DESCARGAR PDF




Documentos relacionados