Comprehensive Evaluation of AmpliSeq Transcriptome, a Novel Targeted Whole Transcriptome RNA Sequencing Methodology for Global Gene Expression Analysis.. Homo sapiens

Background: Whole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. However, RNA-seq carries a few limitations, e.g., the requirement of a significant amount of input RNA and complications led by non-specific mapping of short reads. The Ion AmpliSeqTM Transcriptome Human Gene Expression Kit (AmpliSeq) was recently introduced by Life Technologies as a whole-transcriptome, targeted gene quantification kit to overcome these limitations of RNA-seq.To assess the performance of this new methodology, we performed a comprehensive comparison of AmpliSeq with RNA-seq using two well-established next-generation sequencing platforms (Illumina HiSeq and Ion Torrent Proton). We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Results: Using published data from two standard RNA reference samples, we observed a strong concordance of log2 fold change for all genes when comparing AmpliSeq to Illumina HiSeq (Pearson’s r=0.92) and Ion Torrent Proton (Pearson’s r=0.92). We used ROC, Matthew’s correlation coefficient and RMSD to determine the overall performance characteristics. All three statistical methods demonstrate AmpliSeq as a highly accurate method for differential gene expression analysis. Additionally, for genes with high abundance, AmpliSeq outperforms the two RNA-seq methods. When analyzing four closely related hiPSC-CM lines, we show that both AmpliSeq and RNA-seq capture similar global gene expression patterns consistent with known sources of variations. Conclusions: Our study indicates that AmpliSeq excels in the limiting areas of RNA-seq for gene expression quantification analysis. Thus, AmpliSeq stands as a very sensitive and cost-effective approach for very large scale gene expression analysis and mRNA marker screening with high accuracy. Overall design: Comprehensive, performance evaluation of AmpliSeq Transcriptome to standard whole-transcriptome RNA-sequencing methods for large-scale, genome-wide differential gene expression analysis. We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs).

Background: 全转录组测序（RNA-seq）是开展全转录组基因表达分析的强有力手段。然而，RNA-seq存在若干局限，例如需要大量起始RNA，且短读段的非特异性比对会引发分析复杂性。由Life Technologies近期推出的Ion AmpliSeq™ 转录组人类基因表达试剂盒（AmpliSeq），作为一款全转录组靶向基因定量试剂盒，旨在克服RNA-seq的上述局限。为评估该新型方法的性能，我们依托两套成熟的下一代测序平台（Illumina HiSeq与Ion Torrent Proton），开展了AmpliSeq与RNA-seq的全面比对分析。我们采用了标准参考RNA样本以及源自人类诱导多能干细胞分化心肌细胞（hiPSC-CMs）的RNA样本。Results: 借助两套标准RNA参考样本的公开数据，我们对比AmpliSeq与Illumina HiSeq、Ion Torrent Proton的结果时，观察到所有基因的log2倍数变化均呈现极强的一致性（Pearson相关系数r=0.92）。我们采用受试者工作特征曲线（ROC）、马修斯相关系数以及均方根偏差（RMSD）来评估整体性能特征。三种统计方法均证实，AmpliSeq是一种用于差异基因表达分析的高精度方法。此外，对于高丰度基因，AmpliSeq的表现优于两套RNA-seq方法。在分析四株亲缘关系密切的hiPSC-CM细胞系时，我们发现AmpliSeq与RNA-seq均可捕捉到与已知变异来源相符的相似全局基因表达模式。Conclusions: 本研究表明，在基因表达定量分析中，AmpliSeq可弥补RNA-seq的短板领域。因此，AmpliSeq是一种高灵敏度、低成本且高精度的方案，适用于大规模基因表达分析与mRNA标志物筛选。Overall design: 针对大规模全基因组差异基因表达分析场景，开展AmpliSeq转录组与标准全转录组RNA测序方法的全面性能评估。我们采用了标准参考RNA样本以及源自人类诱导多能干细胞分化心肌细胞（hiPSC-CMs）的RNA样本。