Application Notes: Genotyping

Multiplexed Digital PCR for Non-Invasive Prenatal Trisomy Screening

Background

Aneuploidy is a genetic condition in which a person has missing or extra copies of chromosomes. The most common fetal aneuploidies are trisomies in which one chromosome has an additional copy. Of these, Patau syndrome (trisomy 13), Edwards syndrome (trisomy 18) and Down syndrome (trisomy 21) are the 3 most common types. While the discovery of fetal cell-free DNA (cfDNA) has enabled early detection of trisomies by looking for discrepancies in the mother’s blood, high costs have limited the availability of trisomy screening.

With recent improvements, digital PCR has the potential to become the standard of care for trisomy NIPT by providing accurate quantification, fast turnaround time and lower cost. In this study, we demonstrate the performance of a novel 4-color multiplexing NIPT trisomy test on the Absolute Q dPCR platform to simultaneously detect T13, T18 and T21.

Workflow features enable higher accuracy and sensitivity:

  • High partitioning consistency and low dead volume maximizes sample utilization
  • Digital pooling enables larger volumes of cfDNA to be analyzed across more partitions
  • 4 colors enables multi-target screening in a single reaction
Non-Invasive Prenatal Trisomy Screening
Figure 1. A simple workflow for the 4-color Atila NIPT assay testing on Combinati Absolute Q dPCR System with integrated digital PCR and data analysis.

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4-Color Discrimination of Multi-Allele Single Nucleotide Polymorphisms on the Absolute Q

Background

Single nucleotide variants (SNVs) or single nucleotide polymorphisms (SNPs), have been implicated in many diseases. The detection, quantification, and discrimination of SNPs has a myriad of relevant applications in precision medicine. Furthermore, multiplexing a reaction to identify more than one allele target per reaction maximizes the amount of data obtained from a single sample. With high specificity and absolute quantification capabilities, digital PCR (dPCR) offers a technical advantage over many SNP detection or genotyping methods.

The Absolute Q is a fully integrated 4-color digital PCR platform that automates all steps of a typical dPCR reaction including partitioning, thermal cycling, and data acquisition. The microfluidic array partitioning (MAP) plate provides routine and consistent generation of 20,000 identically sized partitions, dispersing over 95 percent of sample across each dPCR reaction, every time. Unlike many available digital PCR systems, the workflow is identical to qPCR, and generates digital PCR results in as little as 90 minutes.

To demonstrate 4-color optical multiplexing for single nucleotide difference discrimination, a 4-plex assay was designed in collaboration with Integrated DNA Technologies for a set of alleles in the CYP2C19 gene (rs12248560). The cytochrome P450 enzyme mediates the primary metabolism of many drugs. Polymorphisms in this gene alter metabolism of certain drug compounds. The polymorphism rs12248560, an ultra-fast metabolism phenotype, has been linked to more favorable outcomes for breast cancer patients receiving the drug tamoxifen.1

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1. Schroth W; Antoniadou L; Fritz P; Schwab M; Muerdter T; Zanger UM; Simon W; Eichelbaum M; Brauch H; “Breast Cancer Treatment Outcome with Adjuvant Tamoxifen Relative to Patient CYP2D6 and CYP2C19 Genotypes.” Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, U.S. National Library of Medicine, pubmed.ncbi.nlm.nih.gov/18024866/.

Rare Allele Detection and Quantification Using IDT rhAmp SNP Genotyping System

Introduction

Precise and sensitive detection of mutation bearing DNA molecules can be critical to drug selection in cancer treatment. For instance, EGFR is an important monitoring target in the treatment of Non-small Cell Lung Carcinoma (NSCLC). Specifically, the presence of EGFR p.T790M mutation indicates tumor resistance to treatment with tyrosine kinase inhibitors (TKIs).1

Integrated DNA Technologies’ rhAmp SNP Genotyping System utilizes RNase H2-depended PCT (rhPCR), a twoenzyme PCR chemistry, which enables highly precise interrogation of SNPs within challenging genomic regions.2 The Combinati Absolute Q digital PCR (dPCR) system utilizes micro-molded plastic picoliter partitions (Figure 1) instead of oil/water emulsions, thus enabling flexibility to accommodate the rhAmp chemistry. For the first time, the IDT rhAmp assay performance was demonstrated on a micro-chamber array based digital PCR platform.

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  1. Moran C. (2011). Importance of molecular features of nonsmall cell lung cancer for choice of treatment. The American
    journal of pathology, 178(5), 1940–1948. https://doi.org/10.1016/j.
    ajpath.2010.12.057
  2. rhAmp SNP Genotyping System. https://www.idtdna.com/pages/
    products/qpcr-and-pcr/genotyping/rhamp-snp-genotyping

Precise BCR-ABL1 Quantification for Chronic Myeloid Leukemia (CML) Monitoring

Abstract

A novel digital PCR (dPCR) platform combining off-the-shelf reagents, a micro-molded plastic microfluidic consumable with a fully integrated single dPCR instrument was developed to address the needs for routine clinical diagnostics. This new platform offers a simplified workflow that enables: rapid time-to-answer; low potential for cross contamination; minimal sample waste; all within a single integrated instrument. Here we showcase the capability of this fully integrated platform to detect and quantify non-small cell lung carcinoma (NSCLC) rare genetic mutants (EGFR T790M) with precision cell-free DNA (cfDNA) standards. Next, we validated the platform with an established chronic myeloid leukemia (CML) fusion gene (BCR-ABL1) assay down to 0.01% mutant allele frequency to highlight the platform’s utility for precision cancer monitoring. Thirdly, using a juvenile myelomonocytic leukemia (JMML) patient-specific assay we demonstrate the ability to precisely track an individual cancer patient’s response to therapy and show the patient’s achievement of complete molecular remission. These three applications highlight the flexibility and utility of this novel fully integrated dPCR platform that has the potential to transform personalized medicine for cancer recurrence monitoring.

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