Publication Date
5-1-2024
Journal
Current Protocols
DOI
10.1002/cpz1.1041
PMID
38774978
PMCID
PMC11115377
PubMedCentral® Posted Date
5-1-2025
PubMedCentral® Full Text Version
Author MSS
Abstract
The detection, validation, and subsequent interpretation of potentially mosaic single-nucleotide variants (SNV) within next-generation sequencing data remains a challenge in both research and clinical laboratory settings. The ability to identify mosaic variants in high genome coverage sequencing data at levels of 1% or lower underscores the necessity for developing guidelines and best practices to verify these variants orthogonally. Droplet digital PCR (ddPCR) has proven to be a powerful and precise method that allows for the determination of low-level variant fractions within a given sample. Herein we describe two precise ddPCR methods using either a fluorescent TaqMan hydrolysis probe approach or an EvaGreen fluorescent dye protocol. The TaqMan approach relies on two different fluorescent probes (FAM and HEX/VIC) each designed to amplify selectively in only the presence of a single nucleotide change denoting the variant or reference position. The fractional abundance is then calculated to determine the relative quantities of both alleles in the final sample. The EvaGreen protocol relies on two independent reactions with oligonucleotide primers designed with the single nucleotide change denoting the variant at the penultimate position of the primer. The relative amplification efficiency of both primer sets (reference and variant) can be compared, to determine the mosaic level of a given variant. As the cost of high-coverage sequencing continues to decrease, the identification of potentially mosaic variants will also increase. The approaches outlined will allow clinicians and researchers a more precise determination of the true mosaic level of a given variant allowing them to better assess not only its potential pathogenicity but also, its possible recurrence risk when offering genetic counseling to families.
Keywords
Polymorphism, Single Nucleotide, Humans, Polymerase Chain Reaction, Mosaicism, Fluorescent Dyes, High-Throughput Nucleotide Sequencing, Droplet digital PCR (ddPCR), Mosaicism, TaqMan, EvaGreen
Published Open-Access
yes
Recommended Citation
Christopher M Grochowski, Richard A Gibbs, and Harsha Doddapaneni, "Validation of Single-Nucleotide Mosaic Variants Through Droplet Digital PCR" (2024). Faculty, Staff and Students Publications. 6236.
https://digitalcommons.library.tmc.edu/baylor_docs/6236