Key considerations for measuring allelic expression on a genomic scale using high-throughput sequencing.
Key considerations for measuring allelic expression on a genomic scale using high-throughput sequencing.
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DOI：
10.1111/j.1365-294x.2010.04472.x
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发表时间：
2010-03
影响因子：
4.9
通讯作者：
Hartl DL
中科院分区：
文献类型：
--
作者：
Fontanillas P;Landry CR;Wittkopp PJ;Russ C;Gruber JD;Nusbaum C;Hartl DL
基因表达的差异被认为是表型多样性的重要来源，因此，剖析基因表达中自然变异的遗传成分对于理解导致基因表达的进化机制很重要。产妇和父亲等位基因的转录结果直接测量等位基因表达而不是总基因表达调节性的变化。这种方法受测序的成绩单数量以及明确分配单个序列片段的能力的影响在这里使用数学建模和计算机模拟的基础，在此基础上，我们确定了在各种条件下通过高通量测序来准确测量相对等位基因表达的最小测序深度，在某个物种中，每个基因至少需要500-1000个测序读数来测试等位基因失衡，因此，至少五个至少五个最终，使用454个测序来研究表达10 000个基因的基因组需要100万次读取。
Differences in gene expression are thought to be an important source of phenotypic diversity, so dissecting the genetic components of natural variation in gene expression is important for understanding the evolutionary mechanisms that lead to adaptation. Gene expression is a complex trait that, in diploid organisms, results from transcription of both maternal and paternal alleles. Directly measuring allelic expression rather than total gene expression offers greater insight into regulatory variation. The recent emergence of high-throughput sequencing offers an unprecedented opportunity to study allelic transcription at a genomic scale for virtually any species. By sequencing transcript pools derived from heterozygous individuals, estimates of allelic expression can be directly obtained. The statistical power of this approach is influenced by the number of transcripts sequenced and the ability to unambiguously assign individual sequence fragments to specific alleles on the basis of transcribed nucleotide polymorphisms. Here, using mathematical modelling and computer simulations, we determine the minimum sequencing depth required to accurately measure relative allelic expression and detect allelic imbalance via high-throughput sequencing under a variety of conditions. We conclude that, within a species, a minimum of 500–1000 sequencing reads per gene are needed to test for allelic imbalance, and consequently, at least five to 10 millions reads are required for studying a genome expressing 10 000 genes. Finally, using 454 sequencing, we illustrate an application of allelic expression by testing for cis-regulatory divergence between closely related Drosophila species.