Allele-specific expression assays using Solexa
Allele-specific expression assays using Solexa
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DOI：
10.1186/1471-2164-10-422
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发表时间：
2009-09-09
期刊：
影响因子：
4.4
通讯作者：
Nuzhdin, Sergey V.
中科院分区：
文献类型：
--
作者：
Main, Bradley J.;Bickel, Ryan D.;Nuzhdin, Sergey V.
背景：等位基因特异性表达（ASE）测定可用于识别顺式，反式和顺式调节变化。了解表达变化的来源对疾病易感性，表型多样性和适应性具有重要意义。虽然通常在SNP处通过相对荧光来测量ASE，但下一代测序提供了一个机会，可以使用读取计数以准确且高通量的方式测量ASE。只有一条solexa流动环。简而言之，含有已知SNP的感兴趣的转录本富含PCR并进行了条形码以实现多路复用。然后，使用测序计数来估计等位基因特异性表达。为了验证这种方法，我们测量了稀释系列中的等位基因偏差，并发现测得的值和期望值之间的高相关性（r> 0.9，p <0.001）。我们将此方法应用于果蝇simulans父母混合物，F1和intodression中的一组5个基因，发现对于这些基因，可以通过顺式调节变化来解释大多数表达差异。结论：我们提供了一种具有能力的新方法。测量大量测定的ASE，使用Solexa Flowcell的一条车道。这将是一种用于分子和人口遗传学研究的有价值技术，以及对全基因组数据集的验证。
Background: Allele-specific expression (ASE) assays can be used to identify cis, trans, and cis-by-trans regulatory variation. Understanding the source of expression variation has important implications for disease susceptibility, phenotypic diversity, and adaptation. While ASE is commonly measured via relative fluorescence at a SNP, next generation sequencing provides an opportunity to measure ASE in an accurate and high-throughput manner using read counts.Results: We introduce a Solexa-based method to perform large numbers of ASE assays using only a single lane of a Solexa flowcell. In brief, transcripts of interest, which contain a known SNP, are PCR enriched and barcoded to enable multiplexing. Then high-throughput sequencing is used to estimate allele-specific expression using sequencing counts. To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001). We applied this method to a set of 5 genes in a Drosophila simulans parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by cis-regulatory variation.Conclusion: We present a new method with the capacity to measure ASE for large numbers of assays using as little as one lane of a Solexa flowcell. This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets.