Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS).

التفاصيل البيبلوغرافية
العنوان:	Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS).
المؤلفون:	Nachmanson D; Department of Pathology, University of Washington, Seattle, Washington 98195, USA., Lian S; Department of Pathology, University of Washington, Seattle, Washington 98195, USA., Schmidt EK; Department of Pathology, University of Washington, Seattle, Washington 98195, USA., Hipp MJ; Department of Pathology, University of Washington, Seattle, Washington 98195, USA., Baker KT; Department of Pathology, University of Washington, Seattle, Washington 98195, USA., Zhang Y; Department of Pathology, University of Washington, Seattle, Washington 98195, USA., Tretiakova M; Department of Pathology, University of Washington, Seattle, Washington 98195, USA., Loubet-Senear K; Department of Pathology, University of Washington, Seattle, Washington 98195, USA., Kohrn BF; Department of Pathology, University of Washington, Seattle, Washington 98195, USA., Salk JJ; Department of Medicine, Division of Medical Oncology, University of Washington, Seattle, Washington 98195, USA., Kennedy SR; Department of Pathology, University of Washington, Seattle, Washington 98195, USA., Risques RA; Department of Pathology, University of Washington, Seattle, Washington 98195, USA.
المصدر:	Genome research [Genome Res] 2018 Oct; Vol. 28 (10), pp. 1589-1599. Date of Electronic Publication: 2018 Sep 19.
نوع المنشور:	Comparative Study; Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.
اللغة:	English
بيانات الدورية:	Publisher: Cold Spring Harbor Laboratory Press Country of Publication: United States NLM ID: 9518021 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1549-5469 (Electronic) Linking ISSN: 10889051 NLM ISO Abbreviation: Genome Res Subsets: MEDLINE
أسماء مطبوعة:	Original Publication: Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory Press, c1995-
مواضيع طبية MeSH:	CRISPR-Cas Systems, Ovarian Neoplasms/genetics , Sequence Analysis, DNA/methods , Tumor Suppressor Protein p53/genetics, DNA/genetics ; DNA Fragmentation ; Female ; High-Throughput Nucleotide Sequencing ; Humans
مستخلص:	Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment. (© 2018 Nachmanson et al.; Published by Cold Spring Harbor Laboratory Press.)
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معلومات مُعتمدة:	R01 CA160674 United States CA NCI NIH HHS; R01 CA181308 United States CA NCI NIH HHS; T32 CA009515 United States CA NCI NIH HHS; R44 CA221426 United States CA NCI NIH HHS
المشرفين على المادة:	0 (TP53 protein, human) 0 (Tumor Suppressor Protein p53) 9007-49-2 (DNA)
تواريخ الأحداث:	Date Created: 20180921 Date Completed: 20190111 Latest Revision: 20221115
رمز التحديث:	20221213
مُعرف محوري في PubMed:	PMC6169890
DOI:	10.1101/gr.235291.118
PMID:	30232196
قاعدة البيانات:	MEDLINE

الوصف
تدمد:	1549-5469
DOI:	10.1101/gr.235291.118