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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)
Nachmanson, Daniela1; Lian, Shenyi1,3; Schmidt, Elizabeth K.1; Hipp, Michael J.1; Baker, Kathryn T.1; Zhang, Yuezheng1; Tretiakova, Maria1; Loubet-Senear, Kaitlyn1; Kohrn, Brendan F.1; Salk, Jesse J.2,4; Kennedy, Scott R.1; Risques, Rosa Ana1
通讯作者Kennedy, Scott R.(1) ; Risques, Rosa Ana(1)
刊名GENOME RESEARCH
2018-10-01
DOI10.1101/gr.235291.118
28期:10页:1589-1599
收录类别SCI
文章类型Article
WOS标题词Science & Technology
类目[WOS]Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Genetics & Heredity
研究领域[WOS]Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Genetics & Heredity
关键词[WOS]RARE MUTATIONS ; CAPTURE ; CANCER ; CELLS
英文摘要

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.

语种英语
WOS记录号WOS:000446043800014
通讯作者邮箱scottrk@uw.edu ; rrisques@uw.edu
第一作者单位Univ Washington, Dept Pathol, Seattle, WA 98195 USA
通讯作者单位Univ Washington, Dept Pathol, Seattle, WA 98195 USA
ISSN1088-9051
引用统计
文献类型期刊论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/145596
专题北京大学临床肿瘤学院_病理科
作者单位1.TwinStrand Biosci, Seattle, WA 98121 USA;
2.Univ Washington, Dept Pathol, Seattle, WA 98195 USA;
3.Univ Washington, Dept Med, Div Med Oncol, Seattle, WA 98195 USA;
4.Peking Univ Canc Hosp & Inst, Dept Pathol, Key Lab Carcinogenesis & Translat Res, Minist Educ Beijing, Beijing 100142, Peoples R China
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Nachmanson, Daniela,Lian, Shenyi,Schmidt, Elizabeth K.,et al. 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)[J]. GENOME RESEARCH,2018,28(10):1589-1599.
APA Nachmanson, Daniela.,Lian, Shenyi.,Schmidt, Elizabeth K..,Hipp, Michael J..,Baker, Kathryn T..,...&Risques, Rosa Ana.(2018).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).GENOME RESEARCH,28(10),1589-1599.
MLA Nachmanson, Daniela,et al."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)".GENOME RESEARCH 28.10(2018):1589-1599.
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