Academic Journal
IMPACT OF FLUORESCENT DYES ON MUTATIONS IN NEXT GENERATION SEQUENCING LIBRARY GENERATION.
| Τίτλος: | IMPACT OF FLUORESCENT DYES ON MUTATIONS IN NEXT GENERATION SEQUENCING LIBRARY GENERATION. |
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| 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) |
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| 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 |
| ISSN: | 1943-4731 |
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| DOI: | 10.7171/001c.166072 |