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Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery
Much of the within species genetic variation is in the form of single nucleotide polymorphisms (SNPs), typically detected by whole genome sequencing (WGS) or microarray-based technologies. However, WGS produces mostly uninformative reads that perfectly match the reference, while microarrays require...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909455/ https://www.ncbi.nlm.nih.gov/pubmed/29361139 http://dx.doi.org/10.1093/nar/gky022 |
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author | Aggeli, Dimitra Karas, Vlad O Sinnott-Armstrong, Nicholas A Varghese, Vici Shafer, Robert W Greenleaf, William J Sherlock, Gavin |
author_facet | Aggeli, Dimitra Karas, Vlad O Sinnott-Armstrong, Nicholas A Varghese, Vici Shafer, Robert W Greenleaf, William J Sherlock, Gavin |
author_sort | Aggeli, Dimitra |
collection | PubMed |
description | Much of the within species genetic variation is in the form of single nucleotide polymorphisms (SNPs), typically detected by whole genome sequencing (WGS) or microarray-based technologies. However, WGS produces mostly uninformative reads that perfectly match the reference, while microarrays require genome-specific reagents. We have developed Diff-seq, a sequencing-based mismatch detection assay for SNP discovery without the requirement for specialized nucleic-acid reagents. Diff-seq leverages the Surveyor endonuclease to cleave mismatched DNA molecules that are generated after cross-annealing of a complex pool of DNA fragments. Sequencing libraries enriched for Surveyor-cleaved molecules result in increased coverage at the variant sites. Diff-seq detected all mismatches present in an initial test substrate, with specific enrichment dependent on the identity and context of the variation. Application to viral sequences resulted in increased observation of variant alleles in a biologically relevant context. Diff-Seq has the potential to increase the sensitivity and efficiency of high-throughput sequencing in the detection of variation. |
format | Online Article Text |
id | pubmed-5909455 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-59094552018-04-24 Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery Aggeli, Dimitra Karas, Vlad O Sinnott-Armstrong, Nicholas A Varghese, Vici Shafer, Robert W Greenleaf, William J Sherlock, Gavin Nucleic Acids Res Methods Online Much of the within species genetic variation is in the form of single nucleotide polymorphisms (SNPs), typically detected by whole genome sequencing (WGS) or microarray-based technologies. However, WGS produces mostly uninformative reads that perfectly match the reference, while microarrays require genome-specific reagents. We have developed Diff-seq, a sequencing-based mismatch detection assay for SNP discovery without the requirement for specialized nucleic-acid reagents. Diff-seq leverages the Surveyor endonuclease to cleave mismatched DNA molecules that are generated after cross-annealing of a complex pool of DNA fragments. Sequencing libraries enriched for Surveyor-cleaved molecules result in increased coverage at the variant sites. Diff-seq detected all mismatches present in an initial test substrate, with specific enrichment dependent on the identity and context of the variation. Application to viral sequences resulted in increased observation of variant alleles in a biologically relevant context. Diff-Seq has the potential to increase the sensitivity and efficiency of high-throughput sequencing in the detection of variation. Oxford University Press 2018-04-20 2018-01-19 /pmc/articles/PMC5909455/ /pubmed/29361139 http://dx.doi.org/10.1093/nar/gky022 Text en © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Aggeli, Dimitra Karas, Vlad O Sinnott-Armstrong, Nicholas A Varghese, Vici Shafer, Robert W Greenleaf, William J Sherlock, Gavin Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery |
title | Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery |
title_full | Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery |
title_fullStr | Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery |
title_full_unstemmed | Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery |
title_short | Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery |
title_sort | diff-seq: a high throughput sequencing-based mismatch detection assay for dna variant enrichment and discovery |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909455/ https://www.ncbi.nlm.nih.gov/pubmed/29361139 http://dx.doi.org/10.1093/nar/gky022 |
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