Quantification of genetic variants in bacterial cultures by Sanger sequencing

Abstract

Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify non-neutral mutations by selection or counter- selection, neutral mutations usually require DNA sequencing to be identified in a population, which are normally expensive and time-consuming. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counter- selection. We propose a novel methodology to quantify DNA using Sanger sequencing, that we validated experimentally with specially-engineered plasmids both in vitro and in co-transformed E. coli by and assessing our predictions with qPCR and fluorescence quantifications. The method relies on the alignment of the electropherograms from the query and reference samples, where we quantify the DNA concentration from the amplitude ratio of aligned electropherogram peaks. Our DNA quantification will allow quantifying genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with consistent reduction of costs compared to canonical approaches such as qPCR.