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IMPACT OF FLUORESCENT DYES ON MUTATIONS IN NEXT GENERATION SEQUENCING LIBRARY GENERATION.

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Τίτλος: IMPACT OF FLUORESCENT DYES ON MUTATIONS IN NEXT GENERATION SEQUENCING LIBRARY GENERATION.
Συγγραφείς: Butty V; Biology Massachusetts Institute of Technology.; Biological Engineering Massachusetts Institute of Technology.; Koch Institute for Integrative Cancer Research Massachusetts Institute of Technology., Patel P; NA n6TEC., Green S; Genomics and Microbiome Core Facility Rush University Medical Center., Levine S; Biology Massachusetts Institute of Technology.; Biological Engineering Massachusetts Institute of Technology.; Koch Institute for Integrative Cancer Research Massachusetts Institute of Technology.
Πηγή: Journal of biomolecular techniques : JBT [J Biomol Tech] 2026 Sep 01; Vol. 37 (3), pp. 51-59. Date of Electronic Publication: 2026 Sep 01 (Print Publication: 2026).
Τύπος έκδοσης: Journal Article
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: Association of Biomolecular Resource Facilities Country of Publication: United States NLM ID: 100888641 Publication Model: eCollection Cited Medium: Internet ISSN: 1943-4731 (Electronic) Linking ISSN: 15240215 NLM ISO Abbreviation: J Biomol Tech Subsets: MEDLINE
Imprint Name(s): Original Publication: Santa Fe, NM : Association of Biomolecular Resource Facilities
Ιατρικοί όροι (MeSH): Fluorescent Dyes*/chemistry , High-Throughput Nucleotide Sequencing*/methods , Mutation* , Gene Library*, DNA Mutational Analysis/methods ; Polymerase Chain Reaction
Περίληψη: DNA labelling fluorescent dyes such as ethidium bromide have long been considered to be highly mutagenic during DNA replication. While recent studies have pushed back on this narrative, the intercalative nature of these dyes continues to raise the possibility that these dyes can induce mutations. The iconPCR instrument by n6tec uses fluorescent dyes to measure amplification in real time and to adjust cycling conditions. However, since this use of qPCR is preparative and not analytical, mutations introduced by fluorescent dyes would be propagated into the sequencing reaction. To address the impact of these dyes on downstream analyses, we have performed routine mutation calling as well as mutational signature analysis on samples amplified using the iconPCR in the presence of either SYBR or EvaGreen. Sequence analysis revealed very minimal impacts of dyes on the reactions, largely within the noise regimen with only subtle changes in mutation rates seen. Mutational signature analysis was unable to identify any key signatures assignable to the dyes in either substitutions or indel domains. The mutational impact of intercalating dyes during fluorescence-guided amplification is therefore minimal and can be disregarded in all but the most sensitive NGS applications.
Σχόλια: Update of: bioRxiv. 2026 Apr 29:2026.04.26.720908. doi: 10.64898/2026.04.26.720908.. (PMID: 42094381)
References: Science. 2022 Apr 22;376(6591):. (PMID: 35949260)
PLoS One. 2017 Oct 26;12(10):e0185056. (PMID: 29073143)
Methods Mol Biol. 2018;1783:149-169. (PMID: 29767361)
Mutat Res. 1999 Feb 2;439(1):37-47. (PMID: 10029672)
Cell Rep. 2013 Jan 31;3(1):246-59. (PMID: 23318258)
Nat Cancer. 2020 Feb;1(2):249-263. (PMID: 32118208)
Nucleic Acids Res. 1991 Sep 25;19(18):5079. (PMID: 1923779)
Gigascience. 2021 Feb 16;10(2):. (PMID: 33590861)
Comput Struct Biotechnol J. 2022 Jun 14;20:2978-2985. (PMID: 35782734)
Nat Genet. 2025 May;57(5):1132-1141. (PMID: 40210680)
Bioinformatics. 2011 Nov 1;27(21):2987-93. (PMID: 21903627)
Bioinformatics. 2023 Dec 1;39(12):. (PMID: 38096571)
Nat Biotechnol. 2019 May;37(5):561-566. (PMID: 30936564)
Nucleic Acids Res. 2015 Dec 2;43(21):e143. (PMID: 26187991)
Proc Natl Acad Sci U S A. 1975 Dec;72(12):5135-9. (PMID: 1061098)
Nat Biotechnol. 2016 May;34(5):547-555. (PMID: 27018799)
J Mol Biol. 1967 Jul 14;27(1):87-106. (PMID: 6033613)
Sci Data. 2016 Jun 07;3:160025. (PMID: 27271295)
Genome Biol. 2011;12(2):R18. (PMID: 21338519)
Grant Information: P30 CA014051 United States CA NCI NIH HHS
Contributed Indexing: Keywords: 16S; Library Quantification; Mutation Signature; NGS; qPCR
Substance Nomenclature: 0 (Fluorescent Dyes)
Entry Date(s): Date Created: 20260904 Date Completed: 20260904 Latest Revision: 20260908
Update Code: 20260908
PubMed Central ID: PMC13541211
DOI: 10.7171/001c.166072
PMID: 42694854
Βάση Δεδομένων: MEDLINE
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  Data: IMPACT OF FLUORESCENT DYES ON MUTATIONS IN NEXT GENERATION SEQUENCING LIBRARY GENERATION.
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  Data: <searchLink fieldCode="AU" term="%22Butty+V%22">Butty V</searchLink>; Biology Massachusetts Institute of Technology.; Biological Engineering Massachusetts Institute of Technology.; Koch Institute for Integrative Cancer Research Massachusetts Institute of Technology.<br /><searchLink fieldCode="AU" term="%22Patel+P%22">Patel P</searchLink>; NA n6TEC.<br /><searchLink fieldCode="AU" term="%22Green+S%22">Green S</searchLink>; Genomics and Microbiome Core Facility Rush University Medical Center.<br /><searchLink fieldCode="AU" term="%22Levine+S%22">Levine S</searchLink>; Biology Massachusetts Institute of Technology.; Biological Engineering Massachusetts Institute of Technology.; Koch Institute for Integrative Cancer Research Massachusetts Institute of Technology.
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  Data: <searchLink fieldCode="JN" term="%22100888641%22">Journal of biomolecular techniques : JBT</searchLink> [J Biomol Tech] 2026 Sep 01; Vol. 37 (3), pp. 51-59. <i>Date of Electronic Publication: </i>2026 Sep 01 (<i>Print Publication: </i>2026).
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  Data: <i>Original Publication</i>: Santa Fe, NM : Association of Biomolecular Resource Facilities
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  Data: <searchLink fieldCode="MM" term="%22Fluorescent+Dyes%22">Fluorescent Dyes*</searchLink>/<searchLink fieldCode="MM" term="%22Fluorescent+Dyes+chemistry%22">chemistry</searchLink> <br /><searchLink fieldCode="MM" term="%22High-Throughput+Nucleotide+Sequencing%22">High-Throughput Nucleotide Sequencing*</searchLink>/<searchLink fieldCode="MM" term="%22High-Throughput+Nucleotide+Sequencing+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Mutation%22">Mutation*</searchLink> <br /><searchLink fieldCode="MM" term="%22Gene+Library%22">Gene Library*</searchLink><br /><searchLink fieldCode="MH" term="%22DNA+Mutational+Analysis%22">DNA Mutational Analysis</searchLink>/<searchLink fieldCode="MH" term="%22DNA+Mutational+Analysis+methods%22">methods</searchLink> ; <searchLink fieldCode="MH" term="%22Polymerase+Chain+Reaction%22">Polymerase Chain Reaction</searchLink>
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  Data: DNA labelling fluorescent dyes such as ethidium bromide have long been considered to be highly mutagenic during DNA replication. While recent studies have pushed back on this narrative, the intercalative nature of these dyes continues to raise the possibility that these dyes can induce mutations. The iconPCR instrument by n6tec uses fluorescent dyes to measure amplification in real time and to adjust cycling conditions. However, since this use of qPCR is preparative and not analytical, mutations introduced by fluorescent dyes would be propagated into the sequencing reaction. To address the impact of these dyes on downstream analyses, we have performed routine mutation calling as well as mutational signature analysis on samples amplified using the iconPCR in the presence of either SYBR or EvaGreen. Sequence analysis revealed very minimal impacts of dyes on the reactions, largely within the noise regimen with only subtle changes in mutation rates seen. Mutational signature analysis was unable to identify any key signatures assignable to the dyes in either substitutions or indel domains. The mutational impact of intercalating dyes during fluorescence-guided amplification is therefore minimal and can be disregarded in all but the most sensitive NGS applications.
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  Data: Update of: bioRxiv. 2026 Apr 29:2026.04.26.720908. doi: 10.64898/2026.04.26.720908.. (PMID: <searchLink fieldCode="PM" term="%2242094381%22">42094381)</searchLink>
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  Data: Science. 2022 Apr 22;376(6591):. (PMID: <searchLink fieldCode="PM" term="%2235949260%22">35949260)</searchLink><br />PLoS One. 2017 Oct 26;12(10):e0185056. (PMID: <searchLink fieldCode="PM" term="%2229073143%22">29073143)</searchLink><br />Methods Mol Biol. 2018;1783:149-169. (PMID: <searchLink fieldCode="PM" term="%2229767361%22">29767361)</searchLink><br />Mutat Res. 1999 Feb 2;439(1):37-47. (PMID: <searchLink fieldCode="PM" term="%2210029672%22">10029672)</searchLink><br />Cell Rep. 2013 Jan 31;3(1):246-59. (PMID: <searchLink fieldCode="PM" term="%2223318258%22">23318258)</searchLink><br />Nat Cancer. 2020 Feb;1(2):249-263. (PMID: <searchLink fieldCode="PM" term="%2232118208%22">32118208)</searchLink><br />Nucleic Acids Res. 1991 Sep 25;19(18):5079. (PMID: <searchLink fieldCode="PM" term="%221923779%22">1923779)</searchLink><br />Gigascience. 2021 Feb 16;10(2):. (PMID: <searchLink fieldCode="PM" term="%2233590861%22">33590861)</searchLink><br />Comput Struct Biotechnol J. 2022 Jun 14;20:2978-2985. (PMID: <searchLink fieldCode="PM" term="%2235782734%22">35782734)</searchLink><br />Nat Genet. 2025 May;57(5):1132-1141. (PMID: <searchLink fieldCode="PM" term="%2240210680%22">40210680)</searchLink><br />Bioinformatics. 2011 Nov 1;27(21):2987-93. (PMID: <searchLink fieldCode="PM" term="%2221903627%22">21903627)</searchLink><br />Bioinformatics. 2023 Dec 1;39(12):. (PMID: <searchLink fieldCode="PM" term="%2238096571%22">38096571)</searchLink><br />Nat Biotechnol. 2019 May;37(5):561-566. (PMID: <searchLink fieldCode="PM" term="%2230936564%22">30936564)</searchLink><br />Nucleic Acids Res. 2015 Dec 2;43(21):e143. (PMID: <searchLink fieldCode="PM" term="%2226187991%22">26187991)</searchLink><br />Proc Natl Acad Sci U S A. 1975 Dec;72(12):5135-9. (PMID: <searchLink fieldCode="PM" term="%221061098%22">1061098)</searchLink><br />Nat Biotechnol. 2016 May;34(5):547-555. (PMID: <searchLink fieldCode="PM" term="%2227018799%22">27018799)</searchLink><br />J Mol Biol. 1967 Jul 14;27(1):87-106. (PMID: <searchLink fieldCode="PM" term="%226033613%22">6033613)</searchLink><br />Sci Data. 2016 Jun 07;3:160025. (PMID: <searchLink fieldCode="PM" term="%2227271295%22">27271295)</searchLink><br />Genome Biol. 2011;12(2):R18. (PMID: <searchLink fieldCode="PM" term="%2221338519%22">21338519)</searchLink>
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  Data: P30 CA014051 United States CA NCI NIH HHS
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