Systematic evaluation of RNA-Seq preparation protocol performance
Systematic evaluation of RNA-Seq preparation protocol performance
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DOI：
10.1186/s12864-019-5953-1
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发表时间：
2019-07-11
期刊：
影响因子：
4.4
通讯作者：
Shen, Jianjun
中科院分区：
文献类型：
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作者：
Chao, Hsueh-Ping;Chen, Yueping;Shen, Jianjun
背景：RNA-Seq是目前应用最广泛的全转录组分析工具。有许多商业试剂盒可用于制备RNA-Seq库；然而，目前尚不清楚这些试剂盒在以下方面的表现：1)核糖体RNA去除；2)外显子与内含子序列的读取覆盖率或恢复；3)鉴定差异表达基因（DEGs）；4)长链非编码RNA （lncRNA）检测。在RNA-Seq分析中，了解常用RNA-Seq文库制备协议的优势和局限性是很重要的，因为该技术仍然昂贵且耗时。结果：在本研究中，我们对四种RNA-Seq试剂盒进行了全面评估。我们使用了三种标准输入协议：Illumina TruSeq总RNA和mRNA试剂盒，改进的NuGEN Ovation v2试剂盒和TaKaRa SMARTer Ultra Low RNA试剂盒v3。我们对这些试剂盒的评估包括质量控制措施，如总体可重复性，5 ‘和3 ’端偏倚，以及deg， lncrna和选择性剪接转录物的鉴定。总体而言，我们发现两种Illumina试剂盒在恢复deg方面最相似，并且Illumina，改良的NuGEN和TaKaRa试剂盒允许鉴定相似的deg组。然而，我们也发现Illumina， NuGEN和TaKaRa试剂盒各自富集不同的基因组。结论：在制造商推荐的输入RNA水平下，评估的所有RNA- seq文库制备方案都适用于实验组之间的区分，TruSeq mRNA试剂盒普遍适用于蛋白质编码基因谱的研究。TruSeq方案倾向于捕获高表达和GC含量的基因，而改进的NuGEN方案倾向于捕获较长的基因。在低RNA输入水平下，SMARTer Ultra Low RNA Kit可能是一个不错的选择，尽管在rRNA去除、外显子作图率和恢复的deg方面，它在标准输入水平下不如TruSeq mRNA Kit。因此，RNA-Seq文库制备试剂盒的选择会对数据结果产生深远的影响。因此，它是设计RNA-Seq实验时要考虑的关键参数。
Background: RNA-Seq is currently the most widely used tool to analyze whole-transcriptome profiles. There are numerous commercial kits available to facilitate preparing RNA-Seq libraries; however, it is still not clear how some of these kits perform in terms of: 1) ribosomal RNA removal; 2) read coverage or recovery of exonic vs. intronic sequences; 3) identification of differentially expressed genes (DEGs); and 4) detection of long non-coding RNA (lncRNA). In RNA-Seq analysis, understanding the strengths and limitations of commonly used RNA-Seq library preparation protocols is important, as this technology remains costly and time-consuming.Results: In this study, we present a comprehensive evaluation of four RNA-Seq kits. We used three standard input protocols: Illumina TruSeq Stranded Total RNA and mRNA kits, a modified NuGEN Ovation v2 kit, and the TaKaRa SMARTer Ultra Low RNA Kit v3. Our evaluation of these kits included quality control measures such as overall reproducibility, 5 ' and 3 ' end-bias, and the identification of DEGs, lncRNAs, and alternatively spliced transcripts. Overall, we found that the two Illumina kits were most similar in terms of recovering DEGs, and the Illumina, modified NuGEN, and TaKaRa kits allowed identification of a similar set of DEGs. However, we also discovered that the Illumina, NuGEN and TaKaRa kits each enriched for different sets of genes.Conclusions: At the manufacturers' recommended input RNA levels, all the RNA-Seq library preparation protocols evaluated were suitable for distinguishing between experimental groups, and the TruSeq Stranded mRNA kit was universally applicable to studies focusing on protein-coding gene profiles. The TruSeq protocols tended to capture genes with higher expression and GC content, whereas the modified NuGEN protocol tended to capture longer genes. The SMARTer Ultra Low RNA Kit may be a good choice at the low RNA input level, although it was inferior to the TruSeq mRNA kit at standard input level in terms of rRNA removal, exonic mapping rates and recovered DEGs. Therefore, the choice of RNA-Seq library preparation kit can profoundly affect data outcomes. Consequently, it is a pivotal parameter to consider when designing an RNA-Seq experiment.