Allele-specific expression assays using Solexa.

Area of Science:

• Genomics and Molecular Biology
• Population Genetics
• Gene Expression Regulation

Background:

• Allele-specific expression (ASE) assays identify regulatory variation (cis, trans, cis-by-trans).
• Understanding expression variation impacts disease susceptibility, phenotypic diversity, and adaptation.
• Next-generation sequencing offers accurate, high-throughput ASE measurement via read counts, surpassing traditional fluorescence methods.

Purpose of the Study:

• To develop and validate a Solexa sequencing-based method for performing numerous ASE assays efficiently.
• To enable accurate, high-throughput measurement of allele-specific expression using sequencing read counts.
• To investigate the contribution of cis-regulatory variation to gene expression divergence.

Main Methods:

• Developed a Solexa sequencing method for multiplexed ASE assays using barcoded, PCR-enriched transcripts with known SNPs.
• Employed high-throughput sequencing to quantify allele-specific expression based on sequencing read counts.
• Validated the method using a dilution series, showing high correlation (r>0.9, p<0.001) between measured and expected allelic bias.

Main Results:

• The method enables large-scale ASE assays using a single Solexa flowcell lane.
• Application to Drosophila simulans genes revealed that cis-regulatory variation predominantly explains expression divergence.
• High accuracy was confirmed through dilution series experiments.

Conclusions:

• A novel, high-throughput method for measuring allele-specific expression is presented.
• This technique is valuable for molecular and population genetics studies.
• The method serves as an effective tool for verifying genome-wide datasets.