Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2018
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
_version_ 1783360578519040000
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
work_keys_str_mv AT nachmansondaniela targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds
AT lianshenyi targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds
AT schmidtelizabethk targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds
AT hippmichaelj targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds
AT bakerkathrynt targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds
AT zhangyuezheng targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds
AT tretiakovamaria targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds
AT loubetsenearkaitlyn targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds
AT kohrnbrendanf targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds
AT salkjessej targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds
AT kennedyscottr targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds
AT risquesrosaana targetedgenomefragmentationwithcrisprcas9enablesfastandefficientenrichmentofsmallgenomicregionsandultraaccuratesequencingwithlowdnainputcrisprds