Comparison of RNA-Seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling.
Comparison of RNA-Seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling.
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通过聚（A）捕获，核糖体RNA耗竭和DNA微阵列的RNA-SEQ比较用于表达分析。
DOI：
10.1186/1471-2164-15-419
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发表时间：
2014-06-02
期刊：
影响因子：
4.4
通讯作者：
Perou CM
中科院分区：
文献类型：
--
作者：
Zhao W;He X;Hoadley KA;Parker JS;Hayes DN;Perou CM
RNA测序（RNA-Seq）通常用于转录组分析以及鉴定新的转录本和可变剪接事件。通常，RNA-Seq文库是使用mRNA（mRNA-Seq）的poly（A）富集以去除核糖体RNA（rRNA）从总RNA制备的，然而，该方法未能捕获非poly（A）转录物或部分降解的mRNA。因此，mRNA-Seq方案与来自福尔马林固定和石蜡包埋（FFPE）样品的RNA不兼容。为了解决在FFPE材料上进行RNA-Seq的需求，我们评估了两种不同的文库制备方案，其可以与小RNA片段兼容使用。我们从多个肿瘤中获得配对的新鲜冷冻（FF）和FFPE RNA，并将这些RNA进行不同的基因表达谱分析方法。我们使用：（a）通过微阵列、mRNA-Seq、Ribo-Zero-Seq和DSN-Seq（双链体特异性核酸酶）的FF RNA和（B）通过Ribo-Zero-Seq和DSN-Seq的FFPE RNA测试了11个人乳腺肿瘤样品。我们还使用10个TCGA肿瘤作为验证集进行了这些不同的RNA-Seq方案。来自配对RNA样品的数据显示所有方案中以及FF和FFPE RNA之间的转录物定量的高度一致性。在FF和FFPE中，Ribo-Zero-Seq以与mRNA-Seq相当的效率去除rRNA，并且在基因3′端提供等同或更少的偏倚覆盖。与其中69%的碱基被映射到转录组的mRNA-Seq相比，DSN-Seq和Ribo-Zero-Seq包含显著更少的映射到转录组的读段（20-30%）;在这些RNA-Seq方案中，许多（如果不是大多数）读段映射到内含子区域。在mRNA-Seq中需要大约1400万个读段，在Ribo-Zero-Seq或DSN-Seq中需要4500 - 6500万个读段，以实现与标准Agilent DNA微阵列相同的基因检测水平。我们的研究结果表明，与mRNA-Seq和微阵列相比，Ribo-Zero-Seq提供了等效的rRNA去除效率，覆盖均匀性，基于基因组的映射读取以及始终如一的高质量转录本定量。此外，Ribo-Zero-Seq和DSN-Seq使用FFPE RNA具有一致的转录物定量，表明RNA-Seq可与FFPE衍生的RNA一起用于基因表达谱分析。本文的在线版本（doi：10.1186/1471-2164-15-419）包含补充材料，可供授权用户使用。
RNA sequencing (RNA-Seq) is often used for transcriptome profiling as well as the identification of novel transcripts and alternative splicing events. Typically, RNA-Seq libraries are prepared from total RNA using poly(A) enrichment of the mRNA (mRNA-Seq) to remove ribosomal RNA (rRNA), however, this method fails to capture non-poly(A) transcripts or partially degraded mRNAs. Hence, a mRNA-Seq protocol will not be compatible for use with RNAs coming from Formalin-Fixed and Paraffin-Embedded (FFPE) samples. To address the desire to perform RNA-Seq on FFPE materials, we evaluated two different library preparation protocols that could be compatible for use with small RNA fragments. We obtained paired Fresh Frozen (FF) and FFPE RNAs from multiple tumors and subjected these to different gene expression profiling methods. We tested 11 human breast tumor samples using: (a) FF RNAs by microarray, mRNA-Seq, Ribo-Zero-Seq and DSN-Seq (Duplex-Specific Nuclease) and (b) FFPE RNAs by Ribo-Zero-Seq and DSN-Seq. We also performed these different RNA-Seq protocols using 10 TCGA tumors as a validation set. The data from paired RNA samples showed high concordance in transcript quantification across all protocols and between FF and FFPE RNAs. In both FF and FFPE, Ribo-Zero-Seq removed rRNA with comparable efficiency as mRNA-Seq, and it provided an equivalent or less biased coverage on gene 3′ ends. Compared to mRNA-Seq where 69% of bases were mapped to the transcriptome, DSN-Seq and Ribo-Zero-Seq contained significantly fewer reads mapping to the transcriptome (20-30%); in these RNA-Seq protocols, many if not most reads mapped to intronic regions. Approximately 14 million reads in mRNA-Seq and 45–65 million reads in Ribo-Zero-Seq or DSN-Seq were required to achieve the same gene detection levels as a standard Agilent DNA microarray. Our results demonstrate that compared to mRNA-Seq and microarrays, Ribo-Zero-Seq provides equivalent rRNA removal efficiency, coverage uniformity, genome-based mapped reads, and consistently high quality quantification of transcripts. Moreover, Ribo-Zero-Seq and DSN-Seq have consistent transcript quantification using FFPE RNAs, suggesting that RNA-Seq can be used with FFPE-derived RNAs for gene expression profiling. The online version of this article (doi: 10.1186/1471-2164-15-419) contains supplementary material, which is available to authorized users.
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