Fig. 1: Limitations of short-read sequencing in gene editing analysis.

A Schematic representation illustrating the limitations of traditional short-read amplicon sequencing in detecting genetic modifications beyond small indels. Different possible outcomes of an HDR-based gene editing strategy are depicted. Structural variants (SVs), such as large deletions (LD), chromosomal truncations (CT), and translocations (TL), can remove the regions where PCR primers (black arrows) bind, leading to their exclusion from sequencing analysis. Consequently, these undetected modifications introduce bias in mutation quantification. B Skewed quantification. Failure to account for complex genomic aberrations, e.g. by long-read sequencing, can lead to misleading conclusions. The left panel illustrates two hypothetical editing scenarios, with and without a DNA repair modulator. The right panel demonstrates how failure to detect SVs leads to an overestimation of HDR. As the fraction of undetected SVs increases, so does the miscalculation of editing outcomes, potentially compromising data interpretation in gene editing studies.