Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, 'definetherain'.
Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, 'definetherain'.
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DOI：
10.1016/j.jviromet.2014.02.020
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发表时间：
2014-06
影响因子：
3.1
通讯作者：
Frater J
中科院分区：
文献类型：
--
作者：
Jones M;Williams J;Gärtner K;Phillips R;Hurst J;Frater J
描述了从qPCR到ddPCR的方案调整要求。据报道，在低靶标数量下，ddPCR的灵敏度可能会降低。设计并应用了一种新的生物信息学工具“definetherain”，以提高低输入目标数下的液滴调用。“Definetherain”可在www.definetherain.org.uk免费获得。液滴数字PCR（ddPCR）代表了用于定量DNA模板的常规定量PCR（qPCR）的新的替代平台。然而，ddPCR所提供的灵敏度和重现性的拟议改进尚未得到充分证明，部分原因是阳性和阴性反应之间的界限并不总是明确的。提供的数据证明ddPCR系统对两种试剂浓度的灵敏度以及用于定义阳性和阴性结果的临界值的选择。通过实施k-最近聚类，产生了提高ddPCR准确性的截止值，其中靶DNA以低拷贝数存在，这是ddPCR的关键应用。该方法适用于人白蛋白和HIV-1前病毒DNA ddPCR定量方案。这个工具是用JavaScript编写的，可以在http://www.definetherain.org.uk的Web浏览器中免费获得。使用“definetherain”对原始ddPCR数据的分析进行优化表明，通过其实施可以改善低目标数量检测。对患者样本的进一步应用将有助于定义该方法的临床实用性。
The requirement for protocol adaptation from qPCR to ddPCR is characterised. A potential loss of sensitivity for ddPCR at low target numbers is reported. A new bioinformatic tool ‘definetherain’ to improve droplet calling at low input target numbers is devised and applied. ‘Definetherain’ is made available free of charge and open access at www.definetherain.org.uk. Droplet Digital PCR (ddPCR) represents a new and alternative platform to conventional quantitative-PCR (qPCR) for the quantitation of DNA templates. However, the proposed improvement in sensitivity and reproducibility offered by ddPCR is not yet fully proven, partly because the delineation between positive and negative responses is not always clear. Data are presented demonstrating the sensitivity of the ddPCR system to both reagent concentrations and choice of cut-off for defining positive and negative results. By implementing k-nearest clustering, cut-offs are produced that improve the accuracy of ddPCR where target DNA is present at low copy numbers, a key application of ddPCR. This approach is applied to human albumin and HIV-1 proviral DNA ddPCR quantitative protocols. This tool is coded in JavaScript and has been made available for free in a web browser at http://www.definetherain.org.uk. Optimisation of the analyses of raw ddPCR data using ‘definetherain’ indicates that low target number detection can be improved by its implementation. Further application to patient samples will help define the clinical utility of this approach.
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影响因子：
3.1
作者：
Henrich TJ;Gallien S;Li JZ;Pereyra F;Kuritzkes DR
通讯作者：
Kuritzkes DR
影响因子：
3.7
作者：
Kiselinova M;Pasternak AO;De Spiegelaere W;Vogelaers D;Berkhout B;Vandekerckhove L
通讯作者：
Vandekerckhove L
影响因子：
48
作者：
Hindson, Christopher M.;Chevillet, John R.;Briggs, Hilary A.;Gallichotte, Emily N.;Ruf, Ingrid K.;Hindson, Benjamin J.;Vessella, Robert L.;Tewari, Muneesh
通讯作者：
Tewari, Muneesh
影响因子：
7.4
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.
影响因子：
3.7
作者：
Strain MC;Lada SM;Luong T;Rought SE;Gianella S;Terry VH;Spina CA;Woelk CH;Richman DD
通讯作者：
Richman DD