Ensemble of nucleic acid absolute quantitation modules for copy number variation detection and RNA profiling.
Ensemble of nucleic acid absolute quantitation modules for copy number variation detection and RNA profiling.
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DOI：
10.1038/s41467-022-29487-y
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发表时间：
2022-04-04
影响因子：
16.6
通讯作者：
Zhang DY
中科院分区：
文献类型：
--
作者：
Wu LR;Dai P;Wang MX;Chen SX;Cohen EN;Jayachandran G;Zhang JX;Serrano AV;Xie NG;Ueno NT;Reuben JM;Barcenas CH;Zhang DY
当前特定 DNA 序列绝对定量的黄金标准是液滴数字 PCR (ddPCR)，它已应用于拷贝数变异 (CNV) 检测。然而，ddPCR 中的定量模块数量受到荧光通道的限制，从而由于遵循泊松分布的采样误差而限制了 CNV 的灵敏度。在这里，我们开发了一种基于 PCR 的分子条形码 NGS 方法，即定量扩增子测序 (QASeq)，可实现精确的绝对定量，可扩展至 200 多个定量模块。通过高效地将条形码附加到单个目标分子上，2-plex QASeq 在绝对分子计数定量中表现出比 ddPCR 更高且更一致的转化率。多重 QASeq 提高了 CNV 敏感性，可以自信地区分 2.05 倍性和正常 2.00 倍性。我们将多重 QASeq 应用到转移性 ERBB2+ (HER2+ ) 乳腺癌患者的连续纵向血浆 cfDNA 样本中，寻找与肿瘤进展的关联。我们进一步展示了用于靶向表达谱分析的 RNA QASeq panel。由于荧光通道有限，用于灵敏核酸定量的高度多重 ddPCR 仍然具有挑战性。在此，作者报告了定量扩增子测序 (QASeq)，这是一种基于 PCR 的分子条形码 NGS 方法，与高多重性兼容。
Current gold standard for absolute quantitation of a specific DNA sequence is droplet digital PCR (ddPCR), which has been applied to copy number variation (CNV) detection. However, the number of quantitation modules in ddPCR is limited by fluorescence channels, which thus limits the CNV sensitivity due to sampling error following Poisson distribution. Here we develop a PCR-based molecular barcoding NGS approach, quantitative amplicon sequencing (QASeq), for accurate absolute quantitation scalable to over 200 quantitation modules. By attaching barcodes to individual target molecules with high efficiency, 2-plex QASeq exhibits higher and more consistent conversion yield than ddPCR in absolute molecule count quantitation. Multiplexed QASeq improves CNV sensitivity allowing confident distinguishment of 2.05 ploidy from normal 2.00 ploidy. We apply multiplexed QASeq to serial longitudinal plasma cfDNA samples from patients with metastatic ERBB2+ (HER2+ ) breast cancer seeking association with tumor progression. We further show an RNA QASeq panel for targeted expression profiling. Highly multiplexed ddPCR for sensitive nucleic acid quantitation remains challenging due to limited fluorescence channels. Here the authors report quantitative amplicon sequencing (QASeq), a PCR-based molecular barcoding NGS approach which is compatible with high multiplexing.
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影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
Hindson, Benjamin J.;Ness, Kevin D.;Masquelier, Donald A.;Belgrader, Phillip;Heredia, Nicholas J.;Makarewicz, Anthony J.;Bright, Isaac J.;Lucero, Michael Y.;Hiddessen, Amy L.;Legler, Tina C.;Kitano, Tyler K.;Hodel, Michael R.;Petersen, Jonathan F.;Wyatt, Paul W.;Steenblock, Erin R.;Shah, Pallavi H.;Bousse, Luc J.;Troup, Camille B.;Mellen, Jeffrey C.;Wittmann, Dean K.;Erndt, Nicholas G.;Cauley, Thomas H.;Koehler, Ryan T.;So, Austin P.;Dube, Simant;Rose, Klint A.;Montesclaros, Luz;Wang, Shenglong;Stumbo, David P.;Hodges, Shawn P.;Romine, Steven;Milanovich, Fred P.;White, Helen E.;Regan, John F.;Karlin-Neumann, George A.;Hindson, Christopher M.;Saxonov, Serge;Colston, Bill W.
通讯作者：
Colston, Bill W.