Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome
Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome
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DOI：
10.1093/nar/26.4.967
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发表时间：
1998-02-15
影响因子：
14.9
通讯作者：
Nickerson, DA
中科院分区：
文献类型：
--
作者：
Rieder, MJ;Taylor, SL;Nickerson, DA
诊断性重测序在医学和进化遗传学中起着核心作用。在这份报告中，我们描述了一个过程，应用基于荧光的重测序和一套集成的分析工具，以人类线粒体基因组为模型系统，自动化和简化DNA变异的鉴定。（Phred，一种碱基识别器，和Phrap，一种序列组装器）被应用于评估跨序列的每个碱基识别的质量。通过将组装的序列与参考序列进行比较，自动识别和“标记”潜在的DNA变体，我们还表明，采用Consed程序来显示一组高度注释的参考序列，通过提供包含PCR引物、编码和调控序列以及先前已知的DNA变体的位置信息的可视化数据库，极大地简化了数据分析。在测序的12个基因组中，包括29个新变体在内的378个变体被鉴定为沿着有两个异质性位点，由PolyPhred程序自动检测。总体而言，我们记录了对长片段DNA进行高质量和准确的基于荧光的重新测序的容易性和速度，以及应用新方法自动发现和查看这些序列中的DNA变体。
Diagnostic re-sequencing plays a central role in medical and evolutionary genetics, In this report we describe a process that applies fluorescence-based re-sequencing and an integrated set of analysis tools to automate and simplify the identification of DNA variations using the human mitochondrial genome as a model system, Two programs used in genome sequence analysis (Phred, a base-caller, and Phrap, a sequence assembler) are applied to assess the quality of each base call across the sequence, Potential DNA variants are automatically identified and 'tagged' by comparing the assembled sequence with a reference sequence, We also show that employing the Consed program to display a set of highly annotated reference sequences greatly simplifies data analysis by providing a visual database containing information on the location of the PCR primers, coding and regulatory sequences and previously known DNA variants. Among the 12 genomes sequenced 378 variants including 29 new variants were identified along with two heteroplasmic sites, automatically detected by the PolyPhred program, Overall we document the ease and speed of performing high quality and accurate fluorescence-based re-sequencing on long tracts of DNA as well as the application of new approaches to automatically find and view DNA variants among these sequences.