Officials at Jumpcode Genomics reported the results of a study demonstrating the ability to improve genotyping through CRISPR-Cas9-based in vitro repeat depletion, a novel method for high-throughput genotyping where repetitive fragments are removed prior to genotyping with low-pass sequencing.
The study “CRISPR-Cas9-based repeat depletion for high-throughput genotyping of complex plant genomes,” published in Genome Research, shows how Jumpcode Genomics’ depletion technology can be used to remove repetitive elements from genome sequencing libraries, allowing for more accurate genotyping of coding and regulatory regions, according to Yaron Hakak, PhD, CEO, Jumpcode Genetics.
The project was developed by the Functional Genomics Laboratory directed by Massimo Delledonne, PhD, of the University of Verona in collaboration with the group of Roberto Papa, PhD, of the Polytechnic University of Marche.
“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 because of 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.
Focused sequencing data on single-copy regions
“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),” write the investigators. Our results showed that CRISPR-Cas9-driven repeat depletion focuses sequencing data on single-copy regions, thus improving high-density and genome-wide genotyping in large and repetitive genomes.”
The researchers designed a custom set of 566,766 gRNAs targeting 2.9 Gbp of repeats in the 3.76-Gb genome of lentil (Lens culinaris). This depletion method removed approximately 40% of reads mapping to uninformative repeats and resulted in nearly a 10-fold increase in the number of genotyped bases compared to non-depleted libraries. The researchers also observed an increased genotyping accuracy by rescuing thousands of heterozygous variants that otherwise would be missed due to low coverage.
“This method has the potential to accelerate the genetic analysis of crops and other organisms with genomes where such large repeat content increases the cost and complexity of analysis,” says Marzia Rossato, PhD, co-lead author and associate professor at the University of Verona.
“The implications of this study are far-reaching, as 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,” notes Hakak.
“As the scale and complexity of genetic analysis increases over time, better tools are needed to cut through the noise and help studies maximize useable data. We are excited to see our depletion technology have another meaningful impact in genomics.”