EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics - II

Background Next Generation Sequencing technologies have facilitated differential gene expression analysis through RNA-seq and Tag-seq methods. RNA-seq has biases associated with transcript lengths, lacks uniform coverage of regions in mRNA and requires 10–20 times more reads than a typical Tag-seq. Most existing Tag-seq methods either have biases or not high throughput due to use of restriction enzymes or enzymatic manipulation of 5' ends of mRNA or use of RNA ligations. Results We have developed EXpression Profiling through Randomly Sheared cDNA tag Sequencing (EXPRSS) that employs acoustic waves to randomly shear cDNA and generate sequence tags at a relatively defined position (~150-200 bp) from the 3' end of each mRNA. Implementation of the method was verified through comparative analysis of expression data generated from EXPRSS, NlaIII-DGE and Affymetrix microarray and through qPCR quantification of selected genes. EXPRSS is a strand specific and restriction enzyme independent tag sequencing method that does not require cDNA length-based data transformations. EXPRSS is highly reproducible, is high-throughput and it also reveals alternative polyadenylation and polyadenylated antisense transcripts. It is cost-effective using barcoded multiplexing, avoids the biases of existing SAGE and derivative methods and can reveal polyadenylation position from paired-end sequencing. Conclusions EXPRSS Tag-seq provides sensitive and reliable gene expression data and enables high-throughput expression profiling with relatively simple downstream analysis. Overall design: Five weeks old Arabidopsis (Col-0, ein2-5, jar1-1 and npr1-1) leaf discs treated with water or flg22 and a time course of samples were collected; mRNA profiles were generated by deep sequencing on Illumina GAIIx using EXPRSS protocols.

背景 下一代测序（Next Generation Sequencing，NGS）技术通过RNA测序（RNA-seq）与标签测序（Tag-seq）手段，推动了差异基因表达分析的发展。RNA测序存在与转录本长度相关的偏差，无法实现信使RNA（mRNA）区域的均匀覆盖，且所需测序读段数是常规标签测序的10至20倍。现有多数标签测序方法要么存在偏差，要么因使用限制性内切酶、对mRNA 5'端进行酶促修饰或采用RNA连接反应而无法实现高通量。
结果 本研究开发了基于随机剪切互补DNA（cDNA）标签测序的表达谱分析技术（EXpression Profiling through Randomly Sheared cDNA tag Sequencing, EXPRSS），该技术利用声波随机剪切cDNA，并在每条mRNA 3'端附近约150~200 bp的相对固定位置生成序列标签。通过对EXPRSS、NlaIII-DGE与Affymetrix微阵列生成的表达数据进行比较分析，以及对选定基因进行定量聚合酶链反应（qPCR）验证，证实了该方法的可行性。EXPRSS属于链特异性、无需限制性内切酶的标签测序技术，无需基于cDNA长度的数据转换步骤。该技术具有极高的重复性与高通量特性，还可用于检测可变多聚腺苷酸化事件及带有多聚腺苷酸尾的反义转录本。采用条形码多重标记策略时成本效益优异，可规避现有基因表达串行分析（SAGE）及其衍生方法的偏差，且通过双端测序可获取多聚腺苷酸化位点信息。
结论 EXPRSS标签测序可提供灵敏可靠的基因表达数据，且下游分析流程相对简便，能够支持高通量表达谱分析。
整体设计：选取生长5周的拟南芥（Col-0、ein2-5、jar1-1及npr1-1品系）的叶盘，分别以清水或鞭毛蛋白22肽（flg22）处理，并按时间梯度收集样本；采用EXPRSS实验流程，通过Illumina GAIIx测序平台进行深度测序，以获取mRNA表达谱。