RNASeq: the death Knell of expression arrays?. Homo sapiens

Comparison of Solexa and Affy expression in two human tissues. Ultra high-throughput sequencing has been used recently as an alternative to microarrays for genotyping, analysis of methylation patterns and identification of transcription factor binding sites. Here, we describe an application of the Solexa sequencing platform to the detection and comparison of mRNA expression levels. Our goals were to estimate technical variance associated with Solexa sequencing and to assess its ability to identify differentially expressed genes. To do so, we estimated gene expression differences between liver and kidney RNA samples using multiple sequencing replicates, and compared the sequencing data to results obtained from arrays, using the same RNA samples. We show that the sequencing data are highly replicable, with remarkably little technical variation. Indeed, our analyses suggest it may be sufficient to sequence each mRNA sample only once (i.e., using one lane) to accurately estimate gene expression levels. Moreover, our data suggests that Solexa sequencing is comparable, if not superior, to arrays in facilitating the identiffication of differ­entially expressed genes, while allowing for additional analyses such as detection of low-expressed genes, alternative splice variants, and novel transcripts. Based on our observations, we propose an empirical protocol as well as a statistical framework for the analysis of gene expression using ultra high-throughput sequencing technology. We extracted total RNA from liver and kidney samples of a single human male, purified the poly-A mRNA and sheared it prior to cDNA synthesis. The cDNA was then processed into a library of template molecules suitable for sequencing on the Illumina Genome Analyzer. To assess technical variance within and between runs, we sequenced each sample seven times, split across two runs of the machine. To investigate the effects of cDNA concentration, two different cDNA concentrations were used: 3 pM (five lanes per sample) and 1.5 pM (two lanes per sample). Raw 36bp reads were trimmed to 32bp to select high-quality data from the Solexa instrument

两种人体组织中Solexa测序与Affy基因芯片的表达谱对比。近年来，超高通量测序已作为微阵列（microarray）的替代技术，被广泛应用于基因分型、甲基化模式分析以及转录因子结合位点的鉴定。本研究将Solexa测序平台应用于mRNA表达水平的检测与对比分析，研究目标有二：一是评估Solexa测序相关的技术变异度，二是验证其鉴定差异表达基因的能力。为此，我们针对肝脏与肾脏的RNA样本开展多轮测序重复实验以估算基因表达差异，并利用相同的RNA样本，将测序数据与基因芯片（array）的检测结果进行对比。研究结果表明，测序数据具有极高的可重复性，技术变异度极低。进一步分析显示，仅需对每个mRNA样本进行单次测序（即使用一个测序泳道），即可准确估算基因表达水平。此外，本研究数据表明，尽管Solexa测序未必优于基因芯片，但在助力差异表达基因鉴定方面，二者性能相当；同时Solexa测序还可支持低表达基因检测、可变剪接变体鉴定以及新转录本发现等额外分析。基于上述研究结果，我们提出了一套适用于超高通量测序技术的基因表达分析经验流程与统计框架。我们从一名男性受试者的肝脏与肾脏样本中提取总RNA，纯化得到聚腺苷酸（poly-A）mRNA，并在cDNA合成前对其进行片段化处理。随后将cDNA构建为适用于Illumina基因组分析仪（Illumina Genome Analyzer）的测序模板分子文库。为评估测序运行内与运行间的技术变异度，我们将每个样本的测序实验分至两台仪器运行，共完成7次重复测序。为探究cDNA浓度对实验的影响，我们设置了两种不同的cDNA浓度：3皮摩尔（pM，每样本5个测序泳道）与1.5皮摩尔（pM，每样本2个测序泳道）。我们将Solexa仪器产出的原始36bp读段（reads）修剪至32bp，以筛选高质量测序数据。