High-throughput genotyping enables the large-scale analysis of genetic diversity in population genomics and genome-wide association studies that combine the genotypic and phenotypic characterization of large collections of accessions. Sequencing-based approaches for genotyping are progressively replacing traditional genotyping methods due to the lower ascertainment bias. However, genome-wide genotyping based on sequencing becomes expensive in species with large genomes and a high proportion of repetitive DNA. Here we describe the use of CRISPR-Cas9 technology to deplete repetitive elements in the 3.76-Gb genome of lentil (Lens culinaris), 84% consisting of repeats, thus concentrating the sequencing data on coding and regulatory regions (single-copy regions). We designed a custom set of 566,766 gRNAs targeting 2.9 Gbp of repeats and excluding repetitive regions overlapping annotated genes and putative regulatory elements based on ATAC-seq data. The novel depletion method removed ~40% of reads mapping to repeats, increasing those mapping to single-copy regions by ~2.6-fold. When analyzing 25 million fragments, this repeat-to-single-copy shift in the sequencing data increased the number of genotyped bases of ~10-fold compared to nondepleted libraries. In the same condition, we were also able to identify ~12-fold more genetic variants in the single-copy regions and increased the genotyping accuracy by rescuing thousands of heterozygous variants that otherwise would be missed due to low coverage. The method performed similarly regardless of the multiplexing level, type of library or genotypes, including different cultivars and a closely-related species (L. orientalis). Our results demonstrated that CRISPR-Cas9-driven repeat depletion focuses sequencing data on meaningful genomic regions, thus improving high-density and genome-wide genotyping in large and repetitive genomes.

CRISPR-Cas9-based repeat depletion for the high-throughput genotyping of complex plant genomes / Rossato, Marzia; Marcolungo, Luca; De Antoni, Luca; Lopatriello, Giulia; Bellucci, Elisa; Cortinovis, Gaia; Frascarelli, Giulia; Nanni, Laura; Bitocchi, Elena; Di Vittori, Valerio; Vincenzi, Leonardo; Lucchini, Filippo; Bett, Kirstin E; Remsay, Larissa; Konkin, David; Delledonne, Massimo; Papa, Roberto. - In: GENOME RESEARCH. - ISSN 1088-9051. - 33:5(2023), pp. 787-797. [10.1101/gr.277628.122]

CRISPR-Cas9-based repeat depletion for the high-throughput genotyping of complex plant genomes

Bellucci, Elisa;Frascarelli, Giulia;Nanni, Laura;Bitocchi, Elena;Di Vittori, Valerio;Papa, Roberto
2023-01-01

Abstract

High-throughput genotyping enables the large-scale analysis of genetic diversity in population genomics and genome-wide association studies that combine the genotypic and phenotypic characterization of large collections of accessions. Sequencing-based approaches for genotyping are progressively replacing traditional genotyping methods due to the lower ascertainment bias. However, genome-wide genotyping based on sequencing becomes expensive in species with large genomes and a high proportion of repetitive DNA. Here we describe the use of CRISPR-Cas9 technology to deplete repetitive elements in the 3.76-Gb genome of lentil (Lens culinaris), 84% consisting of repeats, thus concentrating the sequencing data on coding and regulatory regions (single-copy regions). We designed a custom set of 566,766 gRNAs targeting 2.9 Gbp of repeats and excluding repetitive regions overlapping annotated genes and putative regulatory elements based on ATAC-seq data. The novel depletion method removed ~40% of reads mapping to repeats, increasing those mapping to single-copy regions by ~2.6-fold. When analyzing 25 million fragments, this repeat-to-single-copy shift in the sequencing data increased the number of genotyped bases of ~10-fold compared to nondepleted libraries. In the same condition, we were also able to identify ~12-fold more genetic variants in the single-copy regions and increased the genotyping accuracy by rescuing thousands of heterozygous variants that otherwise would be missed due to low coverage. The method performed similarly regardless of the multiplexing level, type of library or genotypes, including different cultivars and a closely-related species (L. orientalis). Our results demonstrated that CRISPR-Cas9-driven repeat depletion focuses sequencing data on meaningful genomic regions, thus improving high-density and genome-wide genotyping in large and repetitive genomes.
2023
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11566/317051
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