Construction of normalized RNA-seq libraries for next-generation sequencing using the crab duplex-specific nuclease.
Construction of normalized RNA-seq libraries for next-generation sequencing using the crab duplex-specific nuclease.
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DOI：
10.1002/0471142727.mb0412s94
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发表时间：
2011-04
影响因子：
--
通讯作者：
Seidman, J G
中科院分区：
文献类型：
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作者：
Christodoulou, Danos C;Gorham, Joshua M;Herman, Daniel S;Seidman, J G
RNA-seq是一种通过对数千万个短DNA片段进行并行测序来研究细胞或组织转录组的方法。然而，基因表达水平的宽动态范围（跨越五个数量级以上）需要相当大的过度测序来表征足够深度的低丰度RNA。在这里，我们描述了一种方法，该方法通过标准化或减少从最丰富的RNA种类到最不丰富的RNA种类的范围来实现低丰度RNA的有效测序。这种标准化是使用一种为生成表达序列标签（EST）库而开发的方法来实现的，该方法使用螃蟹双链体特异性核酸酶并利用DNA退火的动力学。也就是说，使双链cDNA变性，使其部分再退火，并且用蟹足特异性核酸酶消化最快速再退火的最丰富的种类。这一过程大大降低了来自高表达RNA的序列读数的比例，有利于评估转录组的全谱序列和结构。
RNA-seq is a method for studying the transcriptome of cells or tissues by massively-parallel sequencing of tens of millions of short DNA fragments. However, the broad dynamic range of gene expression levels, which span more than five orders of magnitude, necessitates considerable over-sequencing to characterize low-abundance RNAs at sufficient depth. Here, we describe a method that enables efficient sequencing of low-abundance RNAs by normalizing or reducing the range of from the most abundant RNA species to the least abundant RNA species. This normalization is achieved using an approach that was developed for generating expressed sequence tag (EST) libraries that uses the crab duplex-specific nuclease and exploits the kinetics of DNA annealing. That is, double stranded cDNA is denatured, allowed to partially re-anneal and the most abundant species, which re-anneal most rapidly are digested with crab duplex-specific nuclease. This procedure substantially decreases the proportion of sequence reads from highly-expressed RNAs, facilitating assessment of the full spectrum of the sequence and structure of transcriptomes.