Genome-wide analysis of allelic expression imbalance in human primary cells by high-throughput transcriptome resequencing.
Genome-wide analysis of allelic expression imbalance in human primary cells by high-throughput transcriptome resequencing.
复制标题
DOI：
10.1093/hmg/ddp473
复制
发表时间：
2010-01-01
影响因子：
3.5
通讯作者：
Plagnol V
中科院分区：
文献类型：
--
作者：
Heap GA;Yang JH;Downes K;Healy BC;Hunt KA;Bockett N;Franke L;Dubois PC;Mein CA;Dobson RJ;Albert TJ;Rodesch MJ;Clayton DG;Todd JA;van Heel DA;Plagnol V
预计通过基因组缔合（GWA）鉴定的许多与疾病相关的变体都可以调节基因表达（ASE）。 RNA-seq）用于ASE分析和表达定量特质基因座。 CD4+ T细胞（n = 4个个体，激活和未经处理的条件），开发了配对的RNA-seq Alignment和ASE分析的工具。为了测试1371个唯一的转录本，以膨胀错误的发现率，我们在控制这些偏见之后估计为50％。考虑到通过HAPMAP QC的SNP的子集，杂合SNP样本对的4.6％显示出不平衡的证据（p <0.001），我们通过细菌克隆和sanger序列验证了四个发现。在多发性硬化症相关的两个样品RS17824933中，CD6中的多个报告基因Exonic SNP，将GWA发现与基因表达的变化。感兴趣的，因此是研究许多与疾病相关遗传变异的调节作用的方法。
Many disease-associated variants identified by genome-wide association (GWA) studies are expected to regulate gene expression. Allele-specific expression (ASE) quantifies transcription from both haplotypes using individuals heterozygous at tested SNPs. We performed deep human transcriptome-wide resequencing (RNA-seq) for ASE analysis and expression quantitative trait locus discovery. We resequenced double poly(A)-selected RNA from primary CD4+ T cells (n = 4 individuals, both activated and untreated conditions) and developed tools for paired-end RNA-seq alignment and ASE analysis. We generated an average of 20 million uniquely mapping 45 base reads per sample. We obtained sufficient read depth to test 1371 unique transcripts for ASE. Multiple biases inflate the false discovery rate which we estimate to be ∼50% for random SNPs. However, after controlling for these biases and considering the subset of SNPs that pass HapMap QC, 4.6% of heterozygous SNP-sample pairs show evidence of imbalance (P < 0.001). We validated four findings by both bacterial cloning and Sanger sequencing assays. We also found convincing evidence for allelic imbalance at multiple reporter exonic SNPs in CD6 for two samples heterozygous at the multiple sclerosis-associated variant rs17824933, linking GWA findings with variation in gene expression. Finally, we show in CD4+ T cells from a further individual that high-throughput sequencing of genomic DNA and RNA-seq following enrichment for targeted gene sequences by sequence capture methods offers an unbiased means to increase the read depth for transcripts of interest, and therefore a method to investigate the regulatory role of many disease-associated genetic variants.