Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations
Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations
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DOI：
10.1073/pnas.1323732111
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发表时间：
2014-02-04
影响因子：
11.1
通讯作者：
Fodor, Stephen P. A.
中科院分区：
文献类型：
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作者：
Fu, Glenn K.;Xu, Weihong;Fodor, Stephen P. A.
我们提出了一个简单的分子索引方法定量靶向RNA测序，其中感兴趣的mRNA选择性地捕获复杂的cDNA文库和测序，以确定其绝对浓度。cDNA片段被单独标记，使得可以通过文库制备和测序过程从原始样品中追踪每个分子。相同序列的cDNA片段的多个拷贝通过标记变得不同，并且在PCR扩增步骤期间产生的复制克隆可以被鉴定并分配给它们不同的亲本分子。选择性捕获使得能够有效地使用测序进行深度采样和绝对定量稀有或瞬时转录物，否则这些转录物将逃脱标准测序方法的检测。我们还构建了一组合成的条形码化RNA分子，其可以作为对照引入样品制备混合物中并用于监测文库构建的效率。定量靶向测序揭示了标准文库制备中极低的效率，这通过使用合成的条形码化RNA分子进一步证实。这一发现表明，标准的文库制备方法会导致稀有转录本的丢失，并强调了监测文库效率和开发更有效的样品制备方法的必要性。
We present a simple molecular indexing method for quantitative targeted RNA sequencing, in which mRNAs of interest are selectively captured from complex cDNA libraries and sequenced to determine their absolute concentrations. cDNA fragments are individually labeled so that each molecule can be tracked from the original sample through the library preparation and sequencing process. Multiple copies of cDNA fragments of identical sequence become distinct through labeling, and replicate clones created during PCR amplification steps can be identified and assigned to their distinct parent molecules. Selective capture enables efficient use of sequencing for deep sampling and for the absolute quantitation of rare or transient transcripts that would otherwise escape detection by standard sequencing methods. We have also constructed a set of synthetic barcoded RNA molecules, which can be introduced as controls into the sample preparation mix and used to monitor the efficiency of library construction. The quantitative targeted sequencing revealed extremely low efficiency in standard library preparations, which were further confirmed by using synthetic barcoded RNA molecules. This finding shows that standard library preparation methods result in the loss of rare transcripts and highlights the need for monitoring library efficiency and for developing more efficient sample preparation methods.