Multiplexed Digital PCR Reference Gene Measurement for Genomic and Cell-Free DNA Analysis.
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All Authors
Yener, D.
Busby, EJ.
Vandesompele, J.
Wils, G.
Richman, SD.
Wood, HM.
Huggett, JF.
Foy, CA.
Devonshire, AS.
LTHT Author
Richman, Susan
Wood, Henry
Wood, Henry
LTHT Department
NIHR Leeds Biomedical Research Centre
Non Medic
Publication Date
2025
Item Type
Journal Article
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Subject
Subject Headings
Abstract
Precision medicine approaches rely on accurate somatic variant detection, where the DNA input into genomic workflows is a key variable. However, there are no gold standard methods for total DNA quantification. In this study, a pentaplex reference gene panel using digital PCR (dPCR) was developed as a candidate reference method. The multiplex approach was compared between two assay chemistries, applied to healthy donor genomic DNA and plasma cell-free DNA (cfDNA) to measure the ERBB2 (HER2) copy number variation in cancer cell line DNA. The multiplex approach demonstrated robust performance with the two assay chemistries, demonstrating comparable results and a wide dynamic range. Ratios of reference genes were close to the expected 1:1 in healthy samples; however, some small but significant differences (<1.2-fold) were observed in one of the five targets. Expanded relative measurement uncertainty was 12.1-19.8% for healthy gDNA and 9.2-25.2% for cfDNA. The multiplex approach afforded lower measurement uncertainty compared to the use of a single reference for total DNA quantification, which is an advantage for its potential use as a calibration method. It avoided potential biases in the application to CNV quantification of cancer samples, where cancer genome instability may be prominent.
Journal
Cells