Comprehensive Detection of Genes Causing a Phenotype using Phenotype Sequencing and Pathway Analysis

TL;DR

This study introduces a pathway-based sequencing approach that enhances the detection of genetic causes of phenotypes, demonstrated on E. coli metabolic mutants, revealing key gene groups linked to the phenotype with improved sensitivity.

Contribution

The paper presents a novel pathway-phenoseq method combining experimental design and high-throughput sequencing for comprehensive gene detection in phenotype studies.

Findings

Identified five gene groups as significant causes of the phenotype

Method improves detection sensitivity over previous approaches

High-scoring gene groups are linked to phenotype and show positive selection evidence

Abstract

Discovering all the genetic causes of a phenotype is an important goal in functional genomics. In this paper we combine an experimental design for multiple independent detections of the genetic causes of a phenotype, with a high-throughput sequencing analysis that maximizes sensitivity for comprehensively identifying them. Testing this approach on a set of 24 mutant strains generated for a metabolic phenotype with many known genetic causes, we show that this pathway-based phenotype sequencing analysis greatly improves sensitivity of detection compared with previous methods, and reveals a wide range of pathways that can cause this phenotype. We demonstrate our approach on a metabolic re-engineering phenotype, the PEP/OAA metabolic node in E. coli, which is crucial to a substantial number of metabolic pathways and under renewed interest for biofuel research. Out of 2157 mutations in these strains, pathway-phenoseq discriminated just five gene groups (12 genes) as statistically significant causes of the phenotype. Experimentally, these five gene groups, and the next two high-scoring pathway-phenoseq groups, either have a clear connection to the PEP metabolite level or offer an alternative path of producing oxaloacetate (OAA), and thus clearly explain the phenotype. These high-scoring gene groups also show strong evidence of positive selection pressure, compared with strictly neutral selection in the rest of the genome.