Department of Pharmacology, Program in Pharmacogenomics; College of Medicine, The Ohio State University Wexner Medical Center, 5184A Graves Hall, 333 West 10th Avenue, Columbus, OH 43210, USA.
BMC Genomics. 2013 Aug 22;14:571. doi: 10.1186/1471-2164-14-571.
Measuring allelic RNA expression ratios is a powerful approach for detecting cis-acting regulatory variants, RNA editing, loss of heterozygosity in cancer, copy number variation, and allele-specific epigenetic gene silencing. Whole transcriptome RNA sequencing (RNA-Seq) has emerged as a genome-wide tool for identifying allelic expression imbalance (AEI), but numerous factors bias allelic RNA ratio measurements. Here, we compare RNA-Seq allelic ratios measured in nine different human brain regions with a highly sensitive and accurate SNaPshot measure of allelic RNA ratios, identifying factors affecting reliable allelic ratio measurement. Accounting for these factors, we subsequently surveyed the variability of RNA editing across brain regions and across individuals.
We find that RNA-Seq allelic ratios from standard alignment methods correlate poorly with SNaPshot, but applying alternative alignment strategies and correcting for observed biases significantly improves correlations. Deploying these methods on a transcriptome-wide basis in nine brain regions from a single individual, we identified genes with AEI across all regions (SLC1A3, NHP2L1) and many others with region-specific AEI. In dorsolateral prefrontal cortex (DLPFC) tissues from 14 individuals, we found evidence for frequent regulatory variants affecting RNA expression in tens to hundreds of genes, depending on stringency for assigning AEI. Further, we find that the extent and variability of RNA editing is similar across brain regions and across individuals.
These results identify critical factors affecting allelic ratios measured by RNA-Seq and provide a foundation for using this technology to screen allelic RNA expression on a transcriptome-wide basis. Using this technology as a screening tool reveals tens to hundreds of genes harboring frequent functional variants affecting RNA expression in the human brain. With respect to RNA editing, the similarities within and between individuals leads us to conclude that this post-transcriptional process is under heavy regulatory influence to maintain an optimal degree of editing for normal biological function.
测量等位基因 RNA 表达比率是一种强大的方法，可用于检测顺式作用调节变体、RNA 编辑、癌症中的杂合性丢失、拷贝数变异和等位基因特异性表观遗传基因沉默。全转录组 RNA 测序（RNA-Seq）已成为识别等位基因表达失衡（AEI）的全基因组工具，但许多因素会影响等位基因 RNA 比率测量的准确性。在这里，我们比较了 9 个人类大脑区域的 RNA-Seq 等位基因比率与高度敏感和准确的 SNaPshot 等位基因 RNA 比率测量值，确定了影响可靠等位基因比率测量的因素。考虑到这些因素，我们随后调查了大脑区域和个体之间的 RNA 编辑变异性。
我们发现，标准比对方法的 RNA-Seq 等位基因比率与 SNaPshot 的相关性较差，但应用替代比对策略并纠正观察到的偏差可显著提高相关性。我们在单个个体的 9 个大脑区域中基于转录组广泛地应用这些方法，鉴定出了所有区域都具有 AEI 的基因（SLC1A3、NHP2L1）和许多具有区域特异性 AEI 的其他基因。在 14 个个体的背外侧前额叶皮层（DLPFC）组织中，我们发现有证据表明，取决于分配 AEI 的严格程度，有数十个到数百个基因受到影响 RNA 表达的常见调节变体。此外，我们发现 RNA 编辑的程度和可变性在大脑区域和个体之间相似。
这些结果确定了影响 RNA-Seq 测量的等位基因比率的关键因素，并为在全转录组范围内使用该技术筛选等位基因 RNA 表达提供了基础。使用该技术作为筛选工具可揭示数十个到数百个基因，这些基因包含影响人类大脑中 RNA 表达的常见功能变体。就 RNA 编辑而言，个体内部和个体之间的相似性使我们得出结论，该转录后过程受到强烈的调控影响，以维持正常生物学功能的最佳编辑程度。
