Dataset Information

Mechanisms for Complex Chromosomal Insertions.

ABSTRACT: Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, constituting ~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Custom high-density aCGH was performed on 10 individuals with available DNA, and breakpoint junctions were fine-mapped at nucleotide resolution by long-range PCR and DNA sequencing in 6 individuals to glean insights into potential mechanisms of formation. We observed microhomologies and templated insertions at the breakpoint junctions, resembling the breakpoint junction signatures found in complex genomic rearrangements generated by replication-based mechanism(s) with iterative template switches. In addition, we analyzed 5 families with apparently balanced insertion in one parent detected by FISH analysis and found that 3 parents had additional small copy-number variants (CNVs) at one or both sides of the inserting fragments as well as at the inserted sites. We propose that replicative repair can result in interchromosomal complex insertions generated through chromothripsis-like chromoanasynthesis involving two or three chromosomes, and cause a significant fraction of apparently balanced insertions harboring small flanking CNVs.

SUBMITTER: Gu S

PROVIDER: S-EPMC5120786 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Mechanisms for Complex Chromosomal Insertions.

Gu Shen S Gu Shen S Szafranski Przemyslaw P Akdemir Zeynep Coban ZC Yuan Bo B Cooper Mitchell L ML Magriñá Maria A MA Bacino Carlos A CA Lalani Seema R SR Breman Amy M AM Smith Janice L JL Patel Ankita A Song Rodger H RH Bi Weimin W Cheung Sau Wai SW Carvalho Claudia M B CM Stankiewicz Paweł P Lupski James R JR

PLoS genetics 20161123 11

Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, constituting ~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ h ...[more]

PMID: 27880765

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Project description:Over the last 30 years the soil bacterium Agrobacterium tumefaciens has been the workhorse tool for plant genome engineering. Replacement of native tumor-inducing (Ti) plasmid elements with customizable cassettes enabled insertion of a sequence of interest as “Transfer DNA” (T-DNA) into the plant genome of interest. Although these T-DNA transfer mechanisms are well understood, detailed understanding of the structure and epigenomic status of insertion events was limited by current technologies. To fill this gap, we analyzed transgenic Arabidopsis thaliana lines from three widely used collections (SALK, SAIL and WISC) with two single molecule technologies, optical genome mapping and nanopore sequencing. Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements with unexpectedly large sizes ranging from 27 to 236 kilobases. De novo nanopore-based genome assemblies for two heterozygous lines resolved T-DNA structures up to 36 kb and revealed large-scale T-DNA associated translocations and exchange of chromosome arm ends. The multiple internally rearranged nature of T-DNA arrays, consisting of identical T-DNA/backbone concatemers made full assembly even for long nanopore reads impossible. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. This unprecedented nucleotide-level definition of T-DNA insertions enabled the mapping of epigenome data. The SALK_059379 T-DNA insertions were enriched for 24nt siRNAs and contained dense cytosine DNA methylation. Transgene silencing via the RNA directed DNA methylation pathway was confirmed by in planta assays. In contrast, SAIL_232 T-DNA sequence was predominantly targeted by 21/22nt siRNAs, and DNA methylation and silencing was limited to the GUS gene, but not the resistance gene. With the emergence of genome editing technologies that rely on Agrobacterium for gene delivery, this study provides new insights into the structural impact of engineering plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.

Dataset Information

Mechanisms for Complex Chromosomal Insertions.

Publications

Mechanisms for Complex Chromosomal Insertions.

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