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CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions
Complete and accurate identification of genetic variants associated with specific phenotypes can be challenging when there is a high level of genomic divergence between individuals in a study and the corresponding reference genome. We have applied the Cas9-mediated enrichment coupled to nanopore seq...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9863143/ https://www.ncbi.nlm.nih.gov/pubmed/36674592 http://dx.doi.org/10.3390/ijms24021076 |
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author | Lopatriello, Giulia Maestri, Simone Alfano, Massimiliano Papa, Roberto Di Vittori, Valerio De Antoni, Luca Bellucci, Elisa Pieri, Alice Bitocchi, Elena Delledonne, Massimo Rossato, Marzia |
author_facet | Lopatriello, Giulia Maestri, Simone Alfano, Massimiliano Papa, Roberto Di Vittori, Valerio De Antoni, Luca Bellucci, Elisa Pieri, Alice Bitocchi, Elena Delledonne, Massimo Rossato, Marzia |
author_sort | Lopatriello, Giulia |
collection | PubMed |
description | Complete and accurate identification of genetic variants associated with specific phenotypes can be challenging when there is a high level of genomic divergence between individuals in a study and the corresponding reference genome. We have applied the Cas9-mediated enrichment coupled to nanopore sequencing to perform a targeted de novo assembly and accurately reconstruct a genomic region of interest. This approach was used to reconstruct a 250-kbp target region on chromosome 5 of the common bean genome (Phaseolus vulgaris) associated with the shattering phenotype. Comparing a non-shattering cultivar (Midas) with the reference genome revealed many single-nucleotide variants and structural variants in this region. We cut five 50-kbp tiled sub-regions of Midas genomic DNA using Cas9, followed by sequencing on a MinION device and de novo assembly, generating a single contig spanning the whole 250-kbp region. This assembly increased the number of Illumina reads mapping to genes in the region, improving their genotypability for downstream analysis. The Cas9 tiling approach for target enrichment and sequencing is a valuable alternative to whole-genome sequencing for the assembly of ultra-long regions of interest, improving the accuracy of downstream genotype–phenotype association analysis. |
format | Online Article Text |
id | pubmed-9863143 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-98631432023-01-22 CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions Lopatriello, Giulia Maestri, Simone Alfano, Massimiliano Papa, Roberto Di Vittori, Valerio De Antoni, Luca Bellucci, Elisa Pieri, Alice Bitocchi, Elena Delledonne, Massimo Rossato, Marzia Int J Mol Sci Article Complete and accurate identification of genetic variants associated with specific phenotypes can be challenging when there is a high level of genomic divergence between individuals in a study and the corresponding reference genome. We have applied the Cas9-mediated enrichment coupled to nanopore sequencing to perform a targeted de novo assembly and accurately reconstruct a genomic region of interest. This approach was used to reconstruct a 250-kbp target region on chromosome 5 of the common bean genome (Phaseolus vulgaris) associated with the shattering phenotype. Comparing a non-shattering cultivar (Midas) with the reference genome revealed many single-nucleotide variants and structural variants in this region. We cut five 50-kbp tiled sub-regions of Midas genomic DNA using Cas9, followed by sequencing on a MinION device and de novo assembly, generating a single contig spanning the whole 250-kbp region. This assembly increased the number of Illumina reads mapping to genes in the region, improving their genotypability for downstream analysis. The Cas9 tiling approach for target enrichment and sequencing is a valuable alternative to whole-genome sequencing for the assembly of ultra-long regions of interest, improving the accuracy of downstream genotype–phenotype association analysis. MDPI 2023-01-05 /pmc/articles/PMC9863143/ /pubmed/36674592 http://dx.doi.org/10.3390/ijms24021076 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Lopatriello, Giulia Maestri, Simone Alfano, Massimiliano Papa, Roberto Di Vittori, Valerio De Antoni, Luca Bellucci, Elisa Pieri, Alice Bitocchi, Elena Delledonne, Massimo Rossato, Marzia CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions |
title | CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions |
title_full | CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions |
title_fullStr | CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions |
title_full_unstemmed | CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions |
title_short | CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions |
title_sort | crispr/cas9-mediated enrichment coupled to nanopore sequencing provides a valuable tool for the precise reconstruction of large genomic target regions |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9863143/ https://www.ncbi.nlm.nih.gov/pubmed/36674592 http://dx.doi.org/10.3390/ijms24021076 |
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