Comprehensive evaluation of AmpliSeq transcriptome, a novel targeted whole transcriptome RNA sequencing methodology for global gene expression analysis.
Comprehensive evaluation of AmpliSeq transcriptome, a novel targeted whole transcriptome RNA sequencing methodology for global gene expression analysis.
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DOI：
10.1186/s12864-015-2270-1
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发表时间：
2015-12-16
期刊：
影响因子：
4.4
通讯作者：
Broeckel U
中科院分区：
文献类型：
--
作者：
Li W;Turner A;Aggarwal P;Matter A;Storvick E;Arnett DK;Broeckel U
全转录组测序(RNA-seq)是分析全转录组基因表达的有效方法。然而，RNA-SEQ有一些限制，例如，需要大量的输入RNA，以及短读写的非特异性映射导致的复杂性。Ion AmpliSeq™转录人类基因表达试剂盒(AmpliSeq)是生命科技公司最近推出的一种全转录组、靶向基因定量试剂盒，以克服RNASeq的这些局限性。为了评估这种新方法的性能，我们使用两个成熟的下一代测序平台(Illumina HiSeq和Ion Torrent Proton)对AmpliSeq和RNA-Seq进行了全面的比较。我们分析了标准参考RNA样本和从人诱导多能干细胞来源的心肌细胞(hiPSC-CMS)获得的RNA样本。使用已公布的两个标准核糖核酸参考样本的数据，当比较AmpliSeq与Illumina HiSeq(Pearson‘s r = 0.92)和Ion Torrent Proton(Pearson’s r = 0.92)时，我们观察到所有基因的log2倍变化具有很强的一致性。我们使用ROC、Matthew‘s相关系数和RMSD来确定总体性能特征。所有三种统计方法都表明AmpliSeq是一种用于差异基因表达分析的高度准确的方法。此外，对于丰度较高的基因，AmpliSeq的性能优于两种RNA-SEQ方法。在分析四个密切相关的HiPSC-CM系时，我们表明AmpliSeq和RNA-seq都捕获了类似的全球基因表达模式，与已知的变异来源一致。我们的研究表明AmpliSeq在RNA-seq的限制区域更适合于基因表达的定量分析。因此，AmpliSeq是一种非常敏感且经济高效的方法，可用于超大规模的基因表达分析和高准确度的mRNA标记筛选。本文的在线版本(doi：10.1186/s12864-0152270-1)包含补充材料，授权用户可以使用。
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 AmpliSeq™ 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). 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. 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. The online version of this article (doi:10.1186/s12864-015-2270-1) contains supplementary material, which is available to authorized users.