PolyPhred: Automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing
PolyPhred: Automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing
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DOI：
10.1093/nar/25.14.2745
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发表时间：
1997-07-15
影响因子：
14.9
通讯作者：
Taylor, SL
中科院分区：
文献类型：
--
作者：
Nickerson, DA;Tobe, VO;Taylor, SL
基于荧光的测序在识别生物和医学上的DNA多态和突变方面发挥着越来越重要的作用，这种技术在产生简单和复杂基因组的参考序列中的应用也推动了新的计算机程序的开发，以在高通量环境中自动碱基调用(Phred)、序列组装(Phrap)和序列组装编辑(Consed)。在本报告中，我们描述了一种被称为PolyPhred的新计算机程序，该程序通过基于荧光的PCR产物测序自动检测杂合单核苷酸替换的存在，其操作与Phred的使用相结合，Phrap和Consed程序以及这些工具一起生成了一个高通量系统，用于通过大规模基于荧光的重新测序来检测DNA多态和突变。对含有已知DNA变体的序列的分析表明，当用荧光染料标记的引物产生序列时，使用单次通过数据的PolyPhred的准确率为99%，而对于用染料标记的终止子制备的序列，准确率与90%相似。
Fluorescence-based sequencing is playing an increasingly important role in efforts to identify DNA polymorphisms and mutations of biological and medical interest, The application of this technology in generating the reference sequence of simple and complex genomes is also driving the development of new computer programs to automate base calling (Phred), sequence assembly (Phrap) and sequence assembly editing (Consed) in high throughput settings, In this report we describe a new computer program known as PolyPhred that automatically detects the presence of heterozygous single nucleotide substitutions by fluorescence-based sequencing of PCR products, Its operations are integrated with the use of the Phred, Phrap and Consed programs and together these tools generate a high throughput system for detecting DNA polymorphisms and mutations by large scale fluorescence-based resequencing. Analysis of sequences containing known DNA variants demonstrates that the accuracy of PolyPhred with single pass data is >99% when the sequences are generated with fluorescent dye-labeled primers and similar to 90% for those prepared with dye-labeled terminators.