The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis.
The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis.
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DOI：
10.1261/rna.052548.115
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发表时间：
2015-10
期刊：
影响因子：
--
通讯作者：
Brierley I
中科院分区：
文献类型：
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作者：
Chung BY;Hardcastle TJ;Jones JD;Irigoyen N;Firth AE;Baulcombe DC;Brierley I
核糖体分析是一种允许全基因组定量分析翻译的技术，近年来得到了广泛的应用。在这里，我们描述了一个修改后的分析协议和软件包，旨在更广泛地受益于翻译社区的简单性和实用性。该协议适用于包括细胞器在内的多种生物，主要基于之前发表的分析方法，但使用双链体特异性核酸酶（DSN）作为一种方便的、不依赖于物种的方法来减少rRNA污染。我们表明，DSN为基础的耗尽相比，其他常用的rRNA耗尽策略，并介绍了小的偏见。分析方案通常产生高水平的三联体周期性，促进编码序列的检测，包括上游、下游和重叠的开放阅读框（ORF）和蛋白质合成终止期间明显的替代核糖体构象。此外，我们还提供了一个软件包，该软件包提供了一套方法，用于解析来自多个样品的核糖体分析数据，将读取与编码序列对齐，推断替代ORF，并绘制数据的平均和转录特异性方面。还提供了用于以适合于翻译和翻译效率的差异分析的形式提取数据的方法。
Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.