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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...

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Autores principales: Aggeli, Dimitra, Karas, Vlad O, Sinnott-Armstrong, Nicholas A, Varghese, Vici, Shafer, Robert W, Greenleaf, William J, Sherlock, Gavin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2018
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.
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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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