دورية أكاديمية
CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions
| العنوان: | CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions |
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| المؤلفون: | Giulia Lopatriello, Simone Maestri, Massimiliano Alfano, Roberto Papa, Valerio Di Vittori, Luca De Antoni, Elisa Bellucci, Alice Pieri, Elena Bitocchi, Massimo Delledonne, Marzia Rossato |
| المصدر: | International Journal of Molecular Sciences, Vol 24, Iss 2, p 1076 (2023) |
| بيانات النشر: | MDPI AG, 2023. |
| سنة النشر: | 2023 |
| المجموعة: | LCC:Biology (General) LCC:Chemistry |
| مصطلحات موضوعية: | de novo assembly, variant calling, Cas9-tiling enrichment, nanopore sequencing, pod-shattering, Biology (General), QH301-705.5, Chemistry, QD1-999 |
| الوصف: | 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. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 1422-0067 1661-6596 |
| Relation: | https://www.mdpi.com/1422-0067/24/2/1076; https://doaj.org/toc/1661-6596; https://doaj.org/toc/1422-0067 |
| DOI: | 10.3390/ijms24021076 |
| URL الوصول: | https://doaj.org/article/40a1c892360943cb8eb85c66325470d2 |
| رقم الأكسشن: | edsdoj.40a1c892360943cb8eb85c66325470d2 |
| قاعدة البيانات: | Directory of Open Access Journals |
Full Text Finder | تدمد: | 14220067 16616596 |
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| DOI: | 10.3390/ijms24021076 |