RNA-Seq：基础，应用和协议

什么是RNA-Seq？

RNA-seq（RNA-sequencing）是一种可以使用样品中RNA的数量和序列的技术下一代测序（NGS）。它分析了转录组，表明我们DNA中编码的哪些基因在多大程度上打开或关闭。在这里，我们研究了为什么RNA-Seq有用，技术的工作原理以及当今常用的基本协议。¹

This can give researchers vital information about the function of genes. For example, the transcriptome can highlight all the tissues in which a gene of unknown function is turned on, which might indicate what its role is. It also captures information about alternative splicing events (Figure 1), which produce different transcripts from one single gene sequence. These events would not be picked up by DNA sequencing. It can also identify转录后修改在mRNA处理期间发生的，例如聚腺苷酸化和5'限。²

Figure 1:RNA-Seq数据使用的简短读取mRNA，该mRNA不含内含子非编码DNA。然后必须将这些读取与参考基因组对齐。

How does RNA-seq work?

Early RNA-seq techniques used Sanger sequencing technology, a technique that although innovative at the time was also low-throughput and costly. It is only recently, with the advent and proliferation ofNGS技术，我们是否能够充分利用RNA-Seq的潜力。⁵

RNA-Seq工作流有多个步骤，可以大致总结为：

1. RNA提取
2. Reverse transcription into cDNA
3. Adapted ligation
4. 放大
5. 测序
   
   

Once you have obtained your RNA sample for analysis, the first step in the technique involves converting the population of RNA to be sequenced into complimentary DNA (cDNA) fragments (a cDNA library). This is done by reverse transcription and allows the RNA to be put into an NGS workflow. The cDNA is then fragmented, and adapters are added to each end of the fragments. These adapters contain functional elements which permit sequencing, for example, the amplification element (which facilitates clonal amplification of the fragments) and the primary sequencing priming site. Following processes of amplification, size selection, clean-up and quality checking, the cDNA library is then analyzed by NGS, producing short sequences that correspond to all or part of the fragment from which it was derived. The depth to which the library is sequenced varies depending on the purpose for which the output data will be used for. Sequencing may follow either single-end or paired-end sequencing methods. Single-read sequencing is a cheaper and faster technique (for reference, about 1% of the cost of Sanger sequencing) that sequences the cDNA fragments from just one end. Paired-end methods sequence from both ends and are therefore more expensive^6,7但是在后续数据重建方面提供了优势。

A further choice must be made between strand-specific and non-strand-specific protocols. The former method means the information about which DNA strand was transcribed is retained. The value of extra information obtained from strand-specific protocols make them the favorable option.

这些读物将在工作流程结束时有数百万美元，然后可以与参考基因组对齐或组装de novo产生跨转录组的RNA序列图。⁸

RNA-Seq vs微阵列：为什么RNA-Seq被认为是优越的

RNA-Seq被广泛认为优于其他技术，例如微阵列杂交。RNA-Seq备受尊敬的身份有几个原因：

不t limited to genomic sequences –unlike hybridization-based approaches, which may require species-specific probes, RNA-seq can detect transcripts from organisms with previously undetermined genomic sequences. This makes it fundamentally superior for the detection of novel transcripts, SNPs or other alterations.^9,10

低背景信号 -RNA-Seq中使用的cDNA序列可以映射到基因组上的靶向区域，从而易于去除实验噪声。此外，在RNA-Seq实验中，跨杂交或不合标准杂交的问题（可能会困扰微阵列实验）不是问题。

更量化 -相对于阵列上检测到的其他信号，微阵列数据仅作为值显示，而RNA-Seq数据是可量化的。RNA-Seq还避免了微阵列在检测非常高或非常低转录水平的问题上存在的问题。