Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes
Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes
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DOI：
10.1073/pnas.1118018109
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发表时间：
2012-01-24
影响因子：
11.1
通讯作者：
Xie, X. Sunney
中科院分区：
文献类型：
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作者：
Shiroguchi, Katsuyuki;Jia, Tony Z.;Xie, X. Sunney
RNA 测序 (RNA-Seq) 是转录组分析的强大工具，但受到指数 PCR 扩增固有的序列依赖性偏差和低拷贝数不准确的阻碍。我们开发了一种简单的策略来减轻这些并发症，从而实现真正的数字化 RNA 测序。逆转录后，大量过量添加条形码序列，并且几乎每个 cDNA 分子都通过在两端随机附着条形码序列来进行独特标记。 PCR 后，我们应用双端深度测序来读取两个条形码和 cDNA 序列。 RNA 丰度不是计算读数的数量，而是根据给定 cDNA 序列观察到的独特条形码序列的数量来测量。我们优化了条形码，即使存在多个测序错误，也可以明确识别。尽管存在序列依赖性偏差和 PCR 扩增噪声，该方法仍允许以单拷贝分辨率进行计数，并且类似于数字 PCR，但可修改以量化整个转录组。我们展示了大肠杆菌的转录组分析，其定量比传统的 RNA-Seq 更准确且可重复。
RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic to exponential PCR amplification. We developed a simple strategy for mitigating these complications, allowing truly digital RNA-Seq. Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, we applied paired-end deep sequencing to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. We optimized the barcodes to be unambiguously identifiable, even in the presence of multiple sequencing errors. This method allows counting with single-copy resolution despite sequence-dependent bias and PCR-amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome. We demonstrated transcriptome profiling of Escherichia coli with more accurate and reproducible quantification than conventional RNA-Seq.