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タイトル
和文:Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes 
英文:Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes 
著者
和文: K. Shiroguchi, T. Z. Jia, P. A. Sims, X. S. Xie, JIATony Z.  
英文: K. Shiroguchi, T. Z. Jia, P. A. Sims, X. S. Xie, Tony Z Jia.  
言語 English 
掲載誌/書名
和文:Proceedings of the National Academy of Sciences 
英文:Proceedings of the National Academy of Sciences 
巻, 号, ページ Vol. 109    No. 4    pp. 1347-1352
出版年月 2012年1月24日 
出版者
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英文: 
会議名称
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開催地
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公式リンク https://www.pnas.org/content/109/4/1347
 
DOI https://doi.org/10.1073/pnas.1118018109
アブストラクト 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.

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