Applications in ddPCR (Droplet Digital PCR)

Quantifying Fetal DNA in Maternal Blood Plasma by ddPCR Using DNA Methylation.

E. Hall, T. Riel, M. Ramesh, M. Gencoglu, O. Mikhaylichenko, R. Dannebaum, S. Margeridon, M. Herrera. Bio-Rad Laboratories, Pleasanton, CA. Association for Molecular Pathology 2022 Annual Meeting Abstracts. J Mol Diagn 2022, 24:S1 Abstract G072.

Introduction: The proportion of cell-free DNA (cfDNA) circulating in maternal blood that originates from the fetus, the fetal fraction, is an important quality control metric when performing tests on fetal-derived cfDNA. Epigenetic differences produce dissimilar DNA methylation patterns, allowing for leveraging regions of high methylation contrast using methylation-sensitive restriction enzyme (MSRE) digestion to quantify fetal and maternal DNA via droplet digital PCR (ddPCR). This advancement positions ddPCR as a faster and less expensive alternative to next-generation sequencing (NGS) for fetal fraction estimation.

Methods: Assays were designed to target MSRE- compatible regions with high methylation contrast between maternal and fetal cfDNA. Fetal assays targeted sites hypermethylated in fetal cfDNA and maternal assays targeted sites hypermethylated in maternal cfDNA. The assay multiplex was tested against contrived and clinical samples using an in-droplet MSRE-ddPCR workflow. The reaction mix was dropletized to create about 20,000 droplets per 24-μL reaction, thermocycled, and analyzed in a QX ONE instrument. The thermocycling profile included a 45-minute MSRE incubation step prior to PCR amplification. Contrived samples were constructed by spiking DNA-free plasma with micrococcal nuclease-digested DNA from an amniotic fluid cell line ("fetal" component) and a B-lymphocyte cell line ("maternal" component). Clinical samples were remnant diagnostic samples with existing NGS non-invasive prenatal testing results attached. DNA was extracted from all samples with the Apostle MiniMax kit on the KingFisher Flex.

Results: From an initial set of 15 assays, a final five-assay multiplex was produced following amplicon sequencing with NGS and ddPCR screening. Although amplicon sequencing did not completely predict ddPCR performance and non- specific interactions, it was valuable for guiding the final ddPCR screen. The five-assay multiplex, consisting of three fetal assays and two maternal assays, produced an excellent linear response against contrived samples from 0% to 25% fetal fraction (R2 >0.99). Similarly, a high correlation was observed between ddPCR-estimated fetal fraction and NGS fetal fraction for a set of clinical samples (n=6, plus two non- pregnant controls, R2 >0.94).

Conclusions: As an epigenetic trait, DNA methylation is a useful way to discriminate between otherwise highly similar DNA sequences in an efficient and effective manner. Leveraging DNA methylation may be done with minimal impact to the standard ddPCR workflow. The high sensitivity, speed, and direct quantification of ddPCR make it an attractive alternative to NGS for fetal fraction estimation.

Dynamics of Plasma EGFR T790M Mutation in Advanced NSCLC: A Multicenter Study. 

Yang et al. Targeted Oncology. 2019;14:719-728. Published: 06 November 2019. 

Background  Droplet digital polymerase chain reaction (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, a ddPCR assay for the epidermal growth factor receptor (EGFR) p.Thr790Met (T790M) mutation suitable for clinical use remains to be established with analytical and clinical validations.  

Objective  We aimed to develop and validate a new ddPCR assay to quantify the T790M mutation in plasma for monitoring and predicting the progression of advanced non-small-cell lung cancer (NSCLC).  Methods  Specificity of the ddPCR assay was evaluated with genomic DNA samples from healthy individuals. The inter- and intraday variations of the assay were evaluated using mixtures of plasmid DNA containing wild-type EGFR and T790M mutation sequences. We assessed the clinical utility of the T790M assay in a multicenter prospective study in patients with advanced NSCLC receiving tyrosine kinase inhibitor (TKI) treatment by analyzing longitudinal plasma DNA samples.  

Results  We set the criteria for a positive call when the following conditions were satisfied: (1) T790M mutation frequency > 0.098% (3 standard deviations above the background signal); (2) at least two positive droplets in duplicate ddPCR reactions. Among the 62 patients with advanced NSCLC exhibiting resistance to TKI treatment, 15 had one or more serial plasma samples that tested positive for T790M. T790M mutation was detected in the plasma as early as 205 days (median 95 days) before disease progression, determined by imaging analysis. Plasma T790M concentrations also correlated with intervention after disease progression.  

Conclusions We developed a ddPCR assay to quantify the T790M mutation in plasma. Quantification of longitudinal plasma T790M mutation may allow noninvasive assessment of drug resistance and guide follow-up treatment in TKI-treated patients with NSCLC.

Trial Registration Clinical Trials.gov identifier: NCT02804100. 

cfDNA was extracted from 2–6 ml of plasma using an Apostle MiniMax High Efficiency cfDNA Isolation Kit (standard edition) (Apostle, Silicon Valley, CA, USA) according to the manufacturer’s instructions.

Segmental duplication as potential biomarkers for non-invasive prenatal testing of aneuploidies.

Chen et al. EBioMedicine August 11, 2021; https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(21)00328-5/fulltext

We developed a computational program whereby available SD regions can be processed and analyzed efficiently for their potential use as biomarkers of the aneuploidy of interest. For the five common aneuploidies, i.e., trisomy 13, 18, 21, and two sex chromosome aneuploidies, a total of 21,772 candidate SD biomarker sequences together with their corresponding primer/probe sets were generated. The primer/probe sets were tested using a real-time PCR-based multicolour melting curve analysis for simultaneous detection of the five common aneuploidies, and yielded 100% clinical sensitivity and 99.64% specificity when subjected to a clinical evaluation. Following the observations that the SD biomarkers for aneuploidy could be better detected by digital PCR with improved accuracy, we established a noninvasive prenatal testing protocol for trisomy 21 and attained 100% concordance with next generation sequencing.

Our study confirmed that SD regions are preferred biomarkers for aneuploidy detection and in particular SD-based digital PCR could find potential use for NIPT of trisomy. A similar strategy can be applied to other chromosomal abnormality and genetic disorders.

The cfDNA was isolated from 1 mL of each plasma sample by the Apostle MiniMax™ High Efficiency cfDNA Isolation Kit (Apostle Inc, San Jose, CA) in line with the manufacturer's instructions.

Next-Generation Digital Polymerase Chain Reaction: High-Dynamic-Range Single-Molecule DNA Counting via Ultrapartitioning.

 Eleen Y. Shum, Janice H. Lai, Sixing Li, Haeun G. Lee, Jesse Soliman, Vedant K. Raol, Cavina K. Lee, Stephen P.A. Fodor, H. Christina Fan Anal. Chem. 2022, Publication Date:December 12, 2022 https://doi.org/10.1021/acs.analchem.2c03649

(Note: Apostle MiniMax technology is used in this study.) 

ABSTRACT: Digital PCR (dPCR) was first conceived for single-molecule quantitation. However, current dPCR systems often require DNA templates to share partitions due to limited partitioning capacities. Here, we introduce UltraPCR, a next-generation dPCR system where DNA counting is performed in a single-molecule regimen through a 6-log dynamic range using a swift and parallelized workflow. Each UltraPCR reaction is divided into >30 million partitions without microfluidics to achieve single template occupancy. Combined with a unique emulsion chemistry, partitions are optically clear, enabling the use of a three-dimensional imaging technique to rapidly detect DNA-positive partitions. Single-molecule occupancy also allows for more straightforward multiplex assay development due to the absence of partition-specific competition. As a proof of concept, we developed a 222-plex UltraPCR assay and demonstrated its potential use as a rapid, low-cost screening assay for noninvasive prenatal testing for as low as 4% trisomy fraction samples with high precision, accuracy, and reproducibility.

(Methods Section) Cell-free plasma was isolated using the Apostle MiniMax kit (Beckman Coulter) according to the manufacturer’s protocol with an elution volume of 60 μL.