Prakash Satya, Racovita Adrian, Petrucci Teresa, Galizi Roberto, Jaramillo Alfonso
School of Life Sciences, University of Warwick, Coventry, UK.
De Novo Synthetic Biology Lab, I2SysBio, CSIC-University of Valencia, Paterna, Spain.
Biodes Res. 2023 Feb 7;5:0007. doi: 10.34133/bdr.0007. eCollection 2023.
Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify nonneutral mutations by selection or counterselection, the identification of neutral mutations in a heterogeneous population usually requires expensive and time-consuming methods such as quantitative or droplet polymerase chain reaction and high-throughput sequencing. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counterselection. We propose a novel method, which we called qSanger, to quantify DNA using amplitude ratios of aligned electropherogram peaks from mixed Sanger sequencing reads. Plasmids expressing enhanced green fluorescent protein and mCherry fluorescent markers were used to validate qSanger both in vitro and in cotransformed via quantitative polymerase chain reaction and fluorescence quantifications. We show that qSanger allows the quantification of genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with substantial reduction of labor and costs compared to canonical approaches.
诸如突变和重组等基因变异在所有培养的生物体中都会自发产生。虽然可以通过选择或反选择来鉴定非中性突变，但在异质群体中鉴定中性突变通常需要昂贵且耗时的方法，如定量或液滴聚合酶链反应以及高通量测序。在不断变化的环境条件下，强制进行瞬时选择或反选择时，中性突变甚至可能成为主导。我们提出了一种新方法，我们称之为qSanger，通过混合桑格测序读数对齐的电泳图峰的幅度比来定量DNA。使用表达增强型绿色荧光蛋白和mCherry荧光标记的质粒在体外和共转化中通过定量聚合酶链反应和荧光定量来验证qSanger。我们表明，qSanger能够从混合桑格测序读数中定量基因变异，包括单碱基自然多态性或新生突变，与传统方法相比，大大减少了劳动力和成本。
