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Author:
Date:
2015-05-20
[Abstract] Recent advances in DNA sequencing capacity to accurately quantify the copy number of individual variants in a large and diverse population allows in parallel determination of the phenotypic effects caused by each genetic modification. This systematic profiling approach is a combination of forward and reverse genetics, which we refer to as quantitative high-resolution genetics (qHRG). This protocol describes how to determine the relative fitness score of each variant compared to wild type (WT) virus based on its frequency determined by Illumina sequencing. Random mutagenesis techniques will be used to introduce randomization at each codon position of the targeted region, thereby generating a comprehensive input mutant library with substitutions at each position of interest (Qi et al. ...
[摘要] DNA测序能力的最近进展，准确量化大和多样群体中单个变体的拷贝数允许平行测定由每个遗传修饰引起的表型效应。这种系统分析方法是正向和反向遗传学的组合，我们称之为定量高分辨率遗传学（qHRG）。该方案描述了如何基于其通过Illumina测序确定的频率来确定每个变体与野生型（WT）病毒相比的相对适合度评分。随机诱变技术将用于在目标区域的每个密码子位置引入随机化，从而产生在每个感兴趣位置具有取代的综合输入突变文库（Qi等人，2014; Wu 等人，2014a; Wu ，2014b）。选择后，每个选定的文库将通过Illumina配对末端测序进行测序，并且确定每个突变的频率。基于频率的变化，可以用回归分析计算每个突变体的相对适合度分数。
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