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

Ostreococcus tauri 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 O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration appears to be random, occurring mainly at a single locus, and thus confirming the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri 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, and often within the vector sequence itself. From these observations, we speculate that a non-homologous end joining-like mechanism is required 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 ecologically important marine algae.

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

Cells ◽

10.3390/cells10030664 ◽

2021 ◽

Vol 10 (3) ◽

pp. 664

Author(s):

Julie Thomy ◽

Frederic Sanchez ◽

Marta Gut ◽

Fernando Cruz ◽

Tyler Alioto ◽

...

Keyword(s):

Insertional Mutagenesis ◽

Chromosomal Rearrangements ◽

Single Copy ◽

Molecular Techniques ◽

End Joining ◽

Structural Variations ◽

Vector Integration ◽

Ostreococcus Tauri ◽

Unicellular Green Alga ◽

Genomic Studies

Ostreococcus tauri 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 O. tauri 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 O. tauri 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.

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Chemotherapy-related secondary leukemias: A role for DNA repair by error-prone non-homologous end joining in topoisomerase II — Induced chromosomal rearrangements

Gene ◽

10.1016/j.gene.2006.12.006 ◽

2007 ◽

Vol 391 (1-2) ◽

pp. 76-79 ◽

Cited By ~ 13

Author(s):

Omar L. Kantidze ◽

Sergey V. Razin

Keyword(s):

Dna Repair ◽

Topoisomerase Ii ◽

Chromosomal Rearrangements ◽

End Joining ◽

Non Homologous End Joining ◽

Secondary Leukemias

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Suppression of non-homologous end joining does not rescue DNA repair defects in Fanconi anemia patient cells

10.1101/151472 ◽

2017 ◽

Cited By ~ 1

Author(s):

Supawat Thongthip ◽

Brooke A. Conti ◽

Francis P. Lach ◽

Agata Smogorzewska

Keyword(s):

Fanconi Anemia ◽

Chromosomal Abnormalities ◽

Chromosomal Rearrangements ◽

Direct Consequence ◽

End Joining ◽

Fanconi Anemia Patient ◽

Interstrand Crosslink ◽

Ligase Iv ◽

Short Palindromic Repeat ◽

Non Homologous End Joining

ABSTRACTSevere cellular sensitivity and aberrant chromosomal rearrangements in response to DNA interstrand crosslink (ICL) inducing agents are hallmarks of Fanconi anemia (FA) deficient cells. These phenotypes have previously been ascribed to inappropriate activity of non-homologous end joining (NHEJ) rather than a direct consequence of DNA ICL repair defects. Here we used chemical inhibitors, RNAi, and Clusterd Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 to inactivate various components of NHEJ in cells from FA patients. We show that suppression of DNA-PKcs, DNA Ligase IV and 53BP1 is not capable of rescuing ICL-induced proliferation defects and only 53BP1 knockout partially suppresses the chromosomal abnormalities of FA patient cells.

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Chromoanagenesis Event Underlies a de novo Pericentric and Multiple Paracentric Inversions in a Single Chromosome Causing Coffin–Siris Syndrome

Frontiers in Genetics ◽

10.3389/fgene.2021.708348 ◽

2021 ◽

Vol 12 ◽

Author(s):

Christopher M. Grochowski ◽

Ana C. V. Krepischi ◽

Jesper Eisfeldt ◽

Haowei Du ◽

Debora R. Bertola ◽

...

Keyword(s):

De Novo ◽

Chromosomal Rearrangements ◽

Whole Genome Sequencing Data ◽

Mendelian Disease ◽

Derivative Chromosome ◽

Sequencing Data ◽

End Joining ◽

Single Chromosome ◽

Complex Structural ◽

Non Homologous End Joining

Chromoanagenesis is a descriptive term that encompasses classes of catastrophic mutagenic processes that generate localized and complex chromosome rearrangements in both somatic and germline genomes. Herein, we describe a 5-year-old female presenting with a constellation of clinical features consistent with a clinical diagnosis of Coffin–Siris syndrome 1 (CSS1). Initial G-banded karyotyping detected a 90-Mb pericentric and a 47-Mb paracentric inversion on a single chromosome. Subsequent analysis of short-read whole-genome sequencing data and genomic optical mapping revealed additional inversions, all clustered on chromosome 6, one of them disrupting ARID1B for which haploinsufficiency leads to the CSS1 disease trait (MIM:135900). The aggregate structural variant data show that the resolved, the resolved derivative chromosome architecture presents four de novo inversions, one pericentric and three paracentric, involving six breakpoint junctions in what appears to be a shuffling of genomic material on this chromosome. Each junction was resolved to nucleotide-level resolution with mutational signatures suggestive of non-homologous end joining. The disruption of the gene ARID1B is shown to occur between the fourth and fifth exon of the canonical transcript with subsequent qPCR studies confirming a decrease in ARID1B expression in the patient versus healthy controls. Deciphering the underlying genomic architecture of chromosomal rearrangements and complex structural variants may require multiple technologies and can be critical to elucidating the molecular etiology of a patient’s clinical phenotype or resolving unsolved Mendelian disease cases.

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The enrichment of breakpoints in late-replicating chromatin provides novel insights into chromoanagenesis mechanisms

10.1101/2020.07.17.206771 ◽

2020 ◽

Author(s):

Nicolas Chatron ◽

Giuliana Giannuzzi ◽

Pierre-Antoine Rollat-Farnier ◽

Flavie Diguet ◽

Eleonora Porcu ◽

...

Keyword(s):

Selective Pressure ◽

Chromosomal Rearrangements ◽

End Joining ◽

Premature Chromosome Condensation ◽

Complex Chromosomal Rearrangement ◽

Three Samples ◽

Complex Chromosomal Rearrangements ◽

Non Homologous End Joining ◽

Reduced Mobility ◽

Dna Ends

AbstractThe rise of pangenomic molecular assays allowed uncovering complex rearrangements named chromoanagenesis that were hypothesized to result from catastrophic shattering events. Constitutional cases have typically been reported individually preventing identification of common features and uncovering the mechanisms at play. We characterized 20 new chromoanagenesis and discovered yet undescribed features. While literature differentiates chromothripsis and its shattering event repaired through non-homologous end joining from chromoanasynthesis born to aberrant replicative processes, we identified shattered chromosomes repaired through a combination of mechanisms. In particular, three samples present with “rearrangement hubs” comprising a fragmented kilobase-long sequence threaded throughout the rearrangement.To assess the mechanisms at play, we merged our data with those of 20 published constitutional complex chromosomal rearrangement cases. We evaluated if the distribution of their 1032 combined breakpoints was distinctive using bootstrap simulations and found that breakpoints tend to keep away from haplosensitive genes suggesting selective pressure. We then compared their distribution with that of 13,310 and 468 breakpoints of cancer complex chromosomal rearrangements and constitutional simple rearrangement samples, respectively. Both complex rearrangement groups showed breakpoint enrichment in late replicating regions suggesting similar origins for constitutional and cancer cases. Simple rearrangement breakpoints but not complex ones were depleted from lamina-associated domains (LADs), possibly as a consequence of reduced mobility of DNA ends bound to lamina.The enrichment of breakpoints in late-replicating chromatin for both constitutional and cancer chromoanagenesis provides an orthogonal support to the premature chromosome condensation hypothesis that was put forward to explain chromoanagenesis.

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