Combining Nanopore and Illumina Sequencing Permits Detailed Analysis of Insertion Mutations and Structural Variations Produced by PEG-Mediated Transformation in <i>Ostreococcus tauri</i>

• Main Authors: Julie Thomy, Frederic Sanchez, Marta Gut, Fernando Cruz, Tyler Alioto, Gwenael Piganeau, Nigel Grimsley, Sheree Yau
• Format: Article
• Language: English
• Published: MDPI AG 2021-03-01
• Series: Cells
• Subjects: Mamiellophyceae; Chlorophyta; polyethylene glycol; copy number; random insertional mutagenesis; unknown sequences
• Online Access: https://www.mdpi.com/2073-4409/10/3/664

<i>Ostreococcus tauri</i> is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in <i>O. tauri </i>a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration occurred randomly at a single locus in the genome and mainly as a single copy. Thus, we confirmed the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the <i>O. tauri </i>model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci. Furthermore, we often observed variations in the vector sequence itself. From these observations, we speculate that a nonhomologous end-joining-like mechanism is employed during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in this ecologically important marine alga.