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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)
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 hybr...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169890/ https://www.ncbi.nlm.nih.gov/pubmed/30232196 http://dx.doi.org/10.1101/gr.235291.118 |
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author | Nachmanson, Daniela Lian, Shenyi Schmidt, Elizabeth K. Hipp, Michael J. Baker, Kathryn T. Zhang, Yuezheng Tretiakova, Maria Loubet-Senear, Kaitlyn Kohrn, Brendan F. Salk, Jesse J. Kennedy, Scott R. Risques, Rosa Ana |
author_facet | Nachmanson, Daniela Lian, Shenyi Schmidt, Elizabeth K. Hipp, Michael J. Baker, Kathryn T. Zhang, Yuezheng Tretiakova, Maria Loubet-Senear, Kaitlyn Kohrn, Brendan F. Salk, Jesse J. Kennedy, Scott R. Risques, Rosa Ana |
author_sort | Nachmanson, Daniela |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6169890 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-61698902019-04-01 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, Daniela Lian, Shenyi Schmidt, Elizabeth K. Hipp, Michael J. Baker, Kathryn T. Zhang, Yuezheng Tretiakova, Maria Loubet-Senear, Kaitlyn Kohrn, Brendan F. Salk, Jesse J. Kennedy, Scott R. Risques, Rosa Ana Genome Res Method 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. Cold Spring Harbor Laboratory Press 2018-10 /pmc/articles/PMC6169890/ /pubmed/30232196 http://dx.doi.org/10.1101/gr.235291.118 Text en © 2018 Nachmanson et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Method Nachmanson, Daniela Lian, Shenyi Schmidt, Elizabeth K. Hipp, Michael J. Baker, Kathryn T. Zhang, Yuezheng Tretiakova, Maria Loubet-Senear, Kaitlyn Kohrn, Brendan F. Salk, Jesse J. Kennedy, Scott R. Risques, Rosa Ana 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) |
title | 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) |
title_full | 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) |
title_fullStr | 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) |
title_full_unstemmed | 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) |
title_short | 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) |
title_sort | 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) |
topic | Method |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169890/ https://www.ncbi.nlm.nih.gov/pubmed/30232196 http://dx.doi.org/10.1101/gr.235291.118 |
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