Comprehensive analysis of RNA-seq kits for standard, low and ultra-low quantity samples

High-throughput RNA-sequencing has now become the gold standard method for whole-transcriptome gene expression analysis. It is widely used in a number of applications studying various transcriptomes of cells and tissues. It is also being increasingly considered for a number of clinical applications, including expression profiling for diagnostics or alternative transcripts detection. However, RNA sequencing can be challenging in some situations, for instance due to low input quantities or degraded RNA samples. Several protocols have been proposed to overcome some of these challenges, and many are available as commercial kits. Here we perform a comprehensive testing of three recent commercial technologies for RNA-seq library preparation (Truseq, Smarter and Smarter Ultra-Low) on human reference tissue preparations, for standard (1ug), low (100 and 10 ng) and ultra-low (< 1 ng) input quantities, and for mRNA and total RNA, stranded or unstranded. We analyze the results using read quality and alignments metrics, gene detection and differential gene expression metrics. Overall, we show that the Truseq kit performs well at 100 ng input quantity, while the Smarter kit shows degraded performances for 100 and 10 ng input quantities, and that the Smarter Ultra-Low kit performs quite well for input quantities < 1 ng. All the results are discussed in details, and we provide guidelines for the selection of a RNA-seq library preparation kits by biologists. Overall design: RNA-seq profiles of Human Universal Reference Total RNA (Clontech) and Human Brain reference RNA (Ambion) were generated by deep sequencing, in duplicate, using Illumina HiSeq2000 or 2500 sequencers.

高通量RNA测序（High-throughput RNA-sequencing）现已成为全转录组基因表达分析的金标准方法，被广泛应用于诸多针对细胞与组织各类转录组的研究之中，同时也日益被诸多临床应用所采纳，例如用于诊断的表达谱分析或可变转录本检测。不过在部分场景中RNA测序仍存在挑战，比如当起始样本量极低或RNA样品发生降解时。已有多种实验方案被提出以克服部分此类难题，其中诸多方案以商业化试剂盒的形式推出。本研究针对三种新近商业化的RNA-seq文库制备技术（Truseq、Smarter及Smarter Ultra-Low）开展了系统性测试，测试载体为人类参考组织样品，设置了标准（1μg）、低量（100 ng与10 ng）及超低量（<1 ng）三类起始投入量，同时覆盖mRNA与总RNA、链特异性与非链特异性的实验分组。本研究通过测序读段质量与比对评估指标、基因检出率以及差异基因表达分析指标对实验结果进行了全面分析。整体结果显示：Truseq试剂盒在100 ng起始投入量下表现优异；Smarter试剂盒在100 ng与10 ng投入量下性能出现退化；而Smarter Ultra-Low试剂盒则在<1 ng的超低起始投入量下表现良好。所有实验结果均进行了详细讨论，同时为生物学家选择RNA-seq文库制备试剂盒提供了规范化指导方案。整体实验设计：本研究借助Illumina HiSeq2000或2500测序平台，对人类通用参考总RNA（Clontech）及人类大脑参考RNA（Ambion）进行了深度测序，每个样品设置两次生物学重复。