Application Notes: Medium Multiplexity

Testing Wastewater Samples for SARS-CoV-2 Using the Absolute Q

Background

As the COVID-19 pandemic continues to have a lasting global impact, effective methods for monitoring communities for early signs of disease spread are a critical need. Screening wastewater, or sewage, for the presence of SARS-CoV-2 viral RNA can be an effective orthogonal monitoring method in addition to ongoing clinical testing. Wastewater serves as pooled samples from members of a community and enables broad collection of surveillance data – even in areas that have limited access to healthcare or testing facilities. Because natural sewage is highly heterogeneous, a method capable of identifying very rare target RNA from a mixture of non-target nucleic acid molecules is required. While quantitative PCR (qPCR) is the current standard for COVID-19 clinical testing, the resulting data can be highly variable due to inadequate sample dilution or chemical contamination. These challenges have a significant impact on measurements of targets that are of low abundance.

In contrast, digital PCR (dPCR) is aptly suited for detecting SARS-CoV-2 targets from wastewater samples. Using dPCR, scientists divide the sample and assay mixture into a large number of independent small reactions, such that zero or one target molecule is present in any individual reaction. Digital PCR overcomes the problem of variability, reduces the impact of many PCR inhibitors, and eliminates the need for standard curves, thus improving accuracy and confidence in rare target detection.1 Digital PCR has been proven to be a more sensitive method of SARS-CoV-2 detection – producing fewer false negatives and demonstrating better performance for low viral load specimens.2

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  1. Sean C. Taylor et al. Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific Reports. 2017 May 25;7(1):2409.
  2. Dong, Lianhua, et al. “Highly Accurate and Sensitive Diagnostic Detection of SARS-CoV-2 by Digital PCR.” MedRxiv, Cold Spring Harbor Laboratory Press, 1 Jan. 2020.

Testing qPCR positive, negative and inconclusive COVID-19 clinical samples using digital PCR

Background

In response to the global outbreak of coronavirus disease 2019 (COVID-19) there is a high demand for sensitive, accurate and consistent tests. Although RT-qPCR has served as the standard of care diagnostic test for the detection of SARS-CoV-2 infection, RT-dPCR (reverse transcription digital PCR) has recently been shown to outperform the traditional method in terms of sensitivity and accuracy.(1,2)

False negative and questionable negative rates using the current screening methodologies (RT-qPCR) have varied over the course of the pandemic and have been reported to be as high as 20%.(3,4) Because of this, asymptomatic patients are at an elevated risk of unknowingly spreading the disease. In addition to the need for more sensitive screening methods, a technology enabling higher accuracy will be critical for screening in determining more accurate rates of re-infection.

A highly sensitive, orthogonal test method to help resolve questionable negatives will increase overall testing accuracy and may also help reduce community transmission. The Combinati Absolute Q with its industry leading accuracy is ideally suited for the disambiguation of questionable negative test outcomes.

The goal of this study was to compare the results obtained using the CDC RT-qPCR assay with a dPCR test on a series of clinical samples. In collaboration with USC Clinical Laboratories, Molecular Pathology at University of Southern California, nucleic acids extracted  from 19 clinical specimens from individuals who tested negative or were diagnosed with COVID-19 were tested on the Combinati Absolute Q dPCR Platform using the |Q|™ SARS-CoV-2 Triplex Assay.

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  1. Suo T, Liu X, Guo M, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv. March 2020:2020.02.29.20029439. doi:10.1101/2020.02.29.20029439
  2. Dong L, Zhou J, Niu C, et al. Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR. medRxiv. March 2020:2020.03.14.20036129. doi:10.1101/2020.03.14.20036129
  3. Woloshin, Steven, et al. “False Negative Tests for SARS-CoV-2 Infection – Challenges and Implications: NEJM.” New England Journal of Medicine, 22 May 2020, www.nejm.org/doi/full/10.1056/NEJMp2015897.
  4. Yu F, et al., “Quantitative Detection and Viral Load Analysis of SARS-CoV-2in Infected Patients”, Clin Infect Dis, 2020

|Q| SARS-CoV-2 Triplex Assay: Multiplexed 1-step RT-dPCR for Accurate Viral Target Detection

Background

The COVID-19 pandemic has drawn heightened concern, with over eleven million positive SARS-CoV-2 cases confirmed worldwide by July 2020.1 RT-qPCR currently serves as the clinical standard for the diagnosis of COVID-19. However in a recent study, it was demonstrated that digital PCR (dPCR) provided better sensitivity for identifying patients who ultimately were diagnosed with COVID-19.2 Reverse transcription digital PCR (RT-dPCR) is a valuable technique which enables improved consistency and lower limits of detection compared to qPCR. Quantification of extremely rare viral RNA target material is also possible without the need for a comparative standard curve. The Combinati |Q| SARS-CoV-2 Triplex Assay was designed as a single tube solution for SARS-CoV-2 identification and quantification with an integrated control assay for human gDNA. This assay paired with the true 1-step RT-dPCR workflow of the Absolute Q dPCR platform enables integration of sample digitization, reverse transcription, PCR and data collection into a single instrument and can be completed in approximately 80 minutes.

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  1. (WHO), World Health Organization. “Coronavirus Disease (COVID-19) – Situation Report 169.” Coronavirus Disease (COVID-2019) Situation Reports, 7 July 2020, 10:00 CEST, www.who.int/docs/default-source/coronaviruse/situation-reports/20200707-covid-19-sitrep-169.pdf?sfvrsn=c6c69c88_2.
  2. Dong L, Zhou J, Niu C, et al. Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR. medRxiv. March 2020:2020.03.14.20036129. doi:10.1101/2020.03.14.20036129

Combinati |Q| SARS-CoV-2 Triplex Assay Digital PCR Protocol

Introduction

The rapid outbreak of COVID-19 originating in Wuhan, China has mobilized unprecedented response to the pandemic across the globe. Numerous diagnostic tests have been deployed to aid in control of disease spread. Positive control materials are required for assay development and to assess overall consistency. To ensure that COVID-19 tests have consistent limits of detection, accurate quantitative measurement of these control materials is critical.

Highlights

  • The Absolute Q provides best-in-class nucleic acid analysis with a complete 90 minute walk-away dPCR workflow.
  • Linear dynamic range verified down to 10 SARS-CoV-2 copies per reaction volume
  • Absolute quantification of reference material will ensure consistent assay performance and disease reporting
  • Accurate dPCR quantification of SARS-CoV-2 targets will benefit the global pandemic response.

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1-Step Reverse Transcription Digital PCR (RT-dPCR) in Under 2 Hours

Background

Reverse Transcription PCR (RT-PCR) is an important tool that allows the assessment of nucleic acid targets that are present in the form of RNA. It has a wide range of applications including gene expression and detection of RNA viruses. During 1-step RT-PCR, reverse transcription of RNA to cDNA occurs in the same reaction vessel as the PCR, which is especially important for clinical applications as the reduced manual handling improves consistency and reduces time to result. Reverse transcription digital PCR (dPCR) further improves the technique by making quantification of extremely rare target material possible without the need for a comparative standard curve – thus enabling better overall consistency and lower limits of detection. Furthermore, recent data suggests dPCR outperforms qPCR in the detection of viral targets such as the widespread SARS-CoV-2 virus.1

The Combinati Absolute Q is a novel 4-color dPCR platform with a complete workflow identical to qPCR. This system overcomes many challenges presented by current dPCR workflows. For example, dPCR typically requires a minimum of 2 instruments to execute the thermal cycling and data collection steps separately. This increases both the time to answer and hands on time as the user is required to move the samples from one stopping point to the next. The Absolute Q’s unique architecture allows it to handle reagent partitioning, reverse transcription, thermal cycling and data collection all on a single instrument and single consumable, enabling a true 1-step RT-dPCR workflow in under 2 hours. In this technical note, we showcase 1-step RT-dPCR on the Absolute Q using the |Q| SARS-CoV-2 Triplex Assay using an RNA-based reference material.

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Workflow and Materials

The SARS-CoV-2 Triplex Assay was designed using published CDC sequences as a single tube solution for SARS-CoV-2 identification and quantification with an integrated control assay for human gDNA. Using the Exact Diagnostics SARS-CoV-2 control material as input, we prepared the SARS-CoV-2 Triplex Assay according to table 1. In addition to quantification of the RNA-based standard as a proof of concept for 1-step RT-dPCR, human genomic DNA alone and water controls were included.

Table 1. 1-step RT-PCR Reaction mix formula.

*1µL of the control standard was loaded per reaction directly from stock. For negative control using human genomic DNA, 50 nanograms were loaded per reaction. Water was adjusted to accommodate the changes in sample volume.

Absolute Q Workflow

After preparing the dPCR mix, 10µL of the reaction mixture was loaded into the MAP16 plate followed by an overlay of 10µL of isolation buffer. The prepared MAP16 plate was then loaded on the Absolute Q. Table 2 details the thermal cycling protocol for RT-dPCR on the Absolute Q.

Table 2. 1-step RT-PCR parameters on the Absolute Q

Table 2. 1-step RT-PCR parameters on the Absolute Q

Quantification of SARS-CoV-2 Targets
Using Reference Materials

Unlike traditional RT-qPCR, RT-dPCR does not require a standard curve or reference sample to identify and quantify targets. The SARS-CoV-2 standard contains synthetic RNA targets from 5 genes of the novel coronavirus and human genomic DNA, which can be used to validate extraction methods. The Combinati SARS-CoV-2 Triplex assay targets the N1 and N2 gene sequences as well as the human RnaseP gene. As a demonstration of the consistency of one-step RT-dPCR using the control RNA as input, we performed the viral quantitation assay in duplicate across 4 separate instruments for a total of 8 replicates using this material.

We identified all three targets in the sample across 8 replicates and saw very few false positives within negative control reactions. We calculated the average concentration of viral and human targets and the associated standard deviation of the SARS-CoV-2 standard in copies per microliter – N1: 358.1 cp/µL (± 22.5), N2: 333.9 cp/µL (± 17.5) and RnaseP: 323.1 cp/µL (± 18.7). Fewer than one positive partition per dPCR reaction was identified on average across all replicate no template control reactions. For the human male control reactions, the values were: N1: 0.0 cp/µL (± 0.0), N2: 0.3 cp/µL (± 0.8). For water only, no template control reactions, the values were: N1: 0.4 cp/µL (± 0.9), N2: 0.1 cp/µL (± 0.4), RnaseP: 0.0 cp/µL (01770.0). Quantitation results were consistent across all four instruments (Figure 1).

Figure 1. Cross instrument Absolute Q quantification consistency using 1-step RT-dPCR. Data shown are the results of the Combinati SARS-CoV-2 Triplex Probe Assay testing the RNA-based SARS-CoV-2 Standard Control ((Exact Diagnostics) and 50 nanograms of human male control DNA (Promega) as a negative control for viral targets. Reactions were run in duplicate for each control material across four instruments for a total of eight replicates each.

Figure 1. Cross instrument Absolute Q quantification consistency using 1-step RT-dPCR. Data shown are the results of the Combinati SARS-CoV-2 Triplex Probe Assay testing the RNA-based SARS-CoV-2 Standard Control ((Exact Diagnostics) and 50 nanograms of human male control DNA (Promega) as a negative control for viral targets. Reactions were run in duplicate for each control material across four instruments for a total of eight replicates each.

Summary

The Absolute Q dPCR platform and its 1-step RT-dPCR technology have broad implications for characterizing infectious diseases beyond COVID-19. The versatile platform can be adapted to a wide range of nucleic acid detection applications requiring absolute quantification. The Absolute Q simplifies dPCR with best-in-class data consistency, a short sample-to-answer time, and flexible multi-color multiplexing capabilities. Combinati aims to lower the barrier to bring dPCR into the lab to accelerate the response to global public health emergencies such as the COVID-19 pandemic.

References

Dong L, Zhou J, Niu C, et al. Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR. medRxiv. March 2020:2020.03.14.20036129. doi:10.1101/2020.03.14.20036129

MAP16 dPCR Plate Flexibility: Iterative Assay Optimization Using a Single Plate

Highlights

  • Experiment flexibility to use a single consumable up to four times
  • Optimization of time allowed for annealing/extension step for a duplex BCR-ABL1 Assay
  • Maintained partition consistencies for iterative use

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The MAP16 consumable was used four times, using one column per experiment to optimize the annealing/extension time for a FAM/HEX multiplexed assay.

Figure 1. The MAP16 consumable was used four times, using one column per experiment to optimize the annealing/extension time for a FAM/HEX multiplexed assay.

Combinati’s patented Microfluidic Array Partitioning (MAP) technology utilizes fixed microchamber arrays and positive pneumatic pressure to partition reagents and perform digital PCR, instead of using fluid-shearing to generate droplets. Each MAP16 plate consists of a 4-unit by 4-unit grid of dPCR reaction units – each unit containing 20,480 fixed partitions. The plate was designed to enable flexibility – meaning up to 16 samples may be used simultaneously or as few as four units, i.e. one column, can be loaded and run at a time without sacrificing data quality. This flexibility can be useful for applications in which lower throughput for dPCR is desired or iterative assay optimization, demonstrated here, is required.

Method Details

In this tech note, an iterative test was performed on a single MAP16 plate to optimize the time allowed for the extension step of PCR for a BCR-ABL1 assay, which detects a gene fusion present in 95% of chronic myeloid leukemia patients. To showcase experiment flexibility of the consumable for repeat uses, we ran four sequential dPCR runs, modifying the extension step of PCR to be 0, 15, 30 and 45 seconds. We compared the final calculated concentration of target as well as compared the fluorescent intensity across each condition. To evaluate the integrity of the MAP plate across successive runs, we calculated the total number of partitions analyzed per condition.

We selected the BCR-ABL pDNA calibrant (Sigma, Cat:ERMAD623), a plasmid containing target sequences for both BCR-ABL1 and ABL1. ERM(R) certification of this well-characterized reference material ensures reliability and comparability of the results. We used a published duplex assay targeting the BCR-ABL1 (FAM) and ABL-1 (HEX) sequences respectively1, and prepared the assay using the Combinati 2X MasterMix. Each reaction contained a final target of 500 copies/µL. Using one column at a time, four replicates were run, and the concentration of each target was quantified in copies/µL. The reagent mix recipe and the dPCR protocol are described in Table 1.

Table 1. dPCR reagent preparation

Table 1. dPCR reagent preparation

For each partition, both low reagent volume and close proximity to the heated surface contribute to PCR robustness at a variety of extension times. Typically, the suggestion for extension time is approximately one minute per 1000 bases. In this study, we test the performance of the duplex assay at increasing extension time intervals (Table 2). In each of the four successive runs, a different column was utilized to evaluate the effects of changing the time allowed for annealing/extension step, starting at 0 seconds and increasing by 15 seconds with each run (Figure 1).

Table 2. Absolute Q dPCR thermal protocol

Table 2. Absolute Q dPCR thermal protocol

Results

The quantification results for both the BCR-ABL1 (FAM) and ABL1 (HEX) targets across the four different extension times (0 seconds, 15 seconds, 30 seconds, and 45 seconds) are shown in Figure 2, together with the representative 2D scatter plots. Extension times at 15 seconds or longer produced accurate quantification, while extension times of 30 seconds or greater provided the best separation between positive and negative partition clusters.

Figure 2. (A) Concentration of multiplex assay targets in the FAM and HEX channels. Colored bars indicate the various extension time used for each condition. Error bars represent the standard deviation, and mean values are noted at the top of each bar.

Figure 2A. Concentration of multiplex assay targets in the FAM and HEX channels. Colored bars indicate the various extension time used for each condition. Error bars represent the standard deviation, and mean values are noted at the top of each bar.

Figure 2B. Two-dimensional dPCR scatter plot data from a single representative reaction per condition. Extension time used denoted at the top.

Figure 2B. Two-dimensional dPCR scatter plot data from a single representative reaction per condition. Extension time used denoted at the top.

The industry standard “targeted minimum” number of analyzed dPCR partitions is typically 20,000. In addition to consistent quantification across repeated use of the same MAP16 plate, the average total number of partitions analyzed per unit remains well above the targeted minimum at 20,252 (±165) partitions per reaction. Figure 3 denotes the average number of accepted partitions and associated standard deviation for the entire plate used, as well as the average per run. Since each dPCR run for this assay requires 40 cycles of PCR, after the fourth run, the partitions in the last column have been exposed to thermal changes for an aggregate of 160 cycles. Even so, the MAP plate yields consistent numbers of acceptable partitions well above 20,000 per unit even in later runs (Figure 3).

Figure 3. Total partitions accepted for analysis by Combinati |Q| Analysis software for one MAP16 plate run 4 separate times to test the effect of extension time on dPCR assay performance. Results from all 4 runs are shown in the first column, and the results of individual runs are shown in subsequent columns. Each point represents the total partition yield from one dPCR unit.

Figure 3. Total partitions accepted for analysis by Combinati |Q| Analysis software for one MAP16 plate run 4 separate times to test the effect of extension time on dPCR assay performance. Results from all 4 runs are shown in the first column, and the results of individual runs are shown in subsequent columns. Each point represents the total partition yield from one dPCR unit.

Summary

Microfluidic Array Partitioning (MAP) technology enhances dPCR. With a simple workflow and highly consistent performance, the MAP plate enables flexibility in experimental design and optimization of dPCR assay conditions without sacrificing robustness.

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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Applied Biosystems™ TaqMan® Liquid Biopsy Assays for Rare Target Quantification

Introduction

Mutation screening is becoming a standard for evaluating treatment options of patients diagnosed with cancer. Notably, certain hotspot mutations can give valuable insight into efficacy of response to various treatments. For example, mutations such as EGFR p.T790M and KRAS p.G12D, p.G12V and p.G13D indicate potential reduced responsiveness to Tyrosine Kinase Inhibitors (TKIs), whereas PIK3CA mutations such as p.H1047R indicate positive response to PI3K/AKT/mTOR signaling pathway inhibitors. With its unparalleled precision and sensitivity, digital PCR is ideally suited for liquid biopsy applications in which low amounts of relevant mutations exist in the sample.

The Combinati Absolute Q, a novel one-step dPCR technology, was used with the Applied Biosystems™ TaqMan™ Liquid Biopsy dPCR Assays to perform rare target detection for 5 hot-spot cancer mutations in the KRAS, EGFR, and PIK3CA genes. MAFs down to 0.1% were detected among a high background wild-type concentration.

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Workflow and Methods

Titration Series Preparation

Five hot-spot cancer mutations were selected for this study: KRAS p.G12D, KRAS p.G12V, KRAS p.G13D, EGFR p.T790M, and PIK3CA p.H1047R. A series of DNA mixtures were prepared by titrating mutation-bearing plasmids into a high background of wild-type normal gDNA for each of the mutations selected. Each point along the titration series contained 15 nanograms of gDNA, and mutation plasmid targeting final mutation allele frequencies (MAFs) of 10%, 1%%, 0.1%. and 0%.

Absolute Q Digital PCR Workflow for pcr machine

Figure 1. Absolute Q workflow.

Absolute Q Workflow

Using the Absolute Q’s simple workflow, PCR mix was prepared using the Combinati 5X MasterMix and loaded into the microfluidic array partitioning (MAP) plate. Subsequently, 10µL of Combinati Isolation Buffer was overlaid into each well used and gaskets applied across all units of the plate. Finally, the plate was loaded onto the Absolute Q where partitioning, thermal cycling, and data collection were completed on the instrument in approximately 90 minutes.

The thermal cycling parameters for use of the TaqMan Liquid Biopsy dPCR Assays on the Absolute Q are listed in Table 1. For select performance evaluation studies, the extension temperature and thermal dwell times were modified. Modifications are indicated when applicable.

Table 1. Thermal Cycling Parameters on the Combinati Absolute Q.

Table 1. Thermal Cycling Parameters on the Combinati Absolute Q.

Results

Rare Mutation Allele Detection

Detecting circulating tumor DNA for liquid biopsy applications is challenging because the molecules bearing the target of interest are only a small fraction of the total circulating cell free DNA collected in the sample. Liquid biopsy assays must be able to accurately quantify rare, single nucleotide polymorphisms, among high levels of wild-type background DNA with outstanding sensitivity and precision. For this study, five cancer-relevant mutations were selected to demonstrate the high precision and sensitivity on the Absolute Q (Workflow and Methods).

For each assay, the total number of mutation molecules and observed mutation allele fraction (MAF) were calculated for each point of the titration series (10%, 1%, 0.1% and 0%). Figure 2a illustrates 2-dimensional digital PCR data generated on the Absolute Q from one replicate of each titration point of the KRAS p.G12V assay test. As expected, the number of total mutation (FAM channel, denoted in blue) positive partitions decreases with each successive point, reflecting a reduction in KRAS pG12V mutation molecules overall while the wild-type (VIC channel, denoted in red) partitions remain constant. Figure 2b shows the linear relationship observed between the expected and observed MAF across all titration series assays. Using a Pearson correlation test, the total number of observed mutation molecules and calculated MAF for each of the titration series were highly correlated with the expectations for all five assays (R = 1.0, p<0.001).

Figure 2A. Two dimensional scatter-plot representation of individual partition amplitudes in the VIC and FAM channels of the Combinati Absolute Q for the KRAS p.G12V specific assay.

Figure 2A. Two dimensional scatter-plot representation of individual partition amplitudes in the VIC and FAM channels of the Combinati Absolute Q for the KRAS p.G12V specific assay. 

Figure 2B. Results of absolute quantifitacion of mutation molecules across a titration mutation bearing plasmid spiked into a background of 15 nanograms of wild-type human genomic DNA. Titration series points were targeted to be 10%, 1% and 0.1% mutation allele frequency (MAF).

Figure 2B Results of absolute quantifitacion of mutation molecules across a titration mutation bearing plasmid spiked into a background of 15 nanograms of wild-type human genomic DNA. Titration series points were targeted to be 10%, 1% and 0.1% mutation allele frequency (MAF).

5X Digital PCR Mix for Low Concentration Samples

A major challenge for many liquid biopsy applications is detection of ultra-rare variants which exist at an extremely low concentration. Sample input volume alongside workflow dead-volume can inhibit the detection of these rare variants by limiting the total number of accessible molecules possible for input. To address this, Combinati offers a 5X dPCR MasterMix for use with liquid biopsy assays. Use of this formulation enables users to add up to 66% more sample volume to each dPCR reaction than when using a 2X formulation. We compared the performance of the 5X MasterMix against a standard 2X PCR MasterMix – testing each assay’s full titration series. Both formulations demonstrate comparable quantification results across each titration series (Figure 3).

Figure 3. Comparison of 5X Combinati mastermix (purple) and 2Xmastermix (orange) mutation molecule quantification for 5 liquid biopsy assays.

Figure 3. Comparison of 5X Combinati mastermix (purple) and 2Xmastermix (orange) mutation molecule quantification for 5 liquid biopsy assays.

Best-in-class Reagent Partitioning with the MAP16

The Combinati Absolute Q utilizes a novel method of reaction isolation on fixed microfluidic array partitioning (MAP) plates. Without a reliance on emulsion based reagent partitioning, over 90% of the loaded sample is analysed in each unit, improving confidence in rare molecule detection. The MAP16 plate consistently generates approximately 20,000 of uniformly filled micro-reactions with no user interaction. The total number of viable partitions across 5 plates are shown in Figure 4. With the exception of one, each unit had over 20,300 viable partitions with an average of 20,457 (±98) accepted partitions per unit.

Figure 4. Total accepted or viable partitions of each of the 16 units per plate for 5 MAP plates used for the Liquid Biopsy assay testing.

Figure 4. Total accepted or viable partitions of each of the 16 units per plate for 5 MAP plates used for the Liquid Biopsy assay testing.

Figure 5. Comparison of ultra-fast and standard dPCR thermal protocols on Absolute Q.

Figure 5. Comparison of ultra-fast and standard dPCR thermal protocols on Absolute Q.

Short Dwell Times Maintain High Specificity

The Absolute Q dPCR platform paired with the MAP plate enables targeted thermal points to be reached much quicker, due to the low reagent volume sequestered in each partition. Using this feature, the dwell times at denaturation and annealing extension were set to 0 seconds, thereby reducing the overall time to complete PCR. To demonstrate heightened precision possible using ultra-fast PCR, we compared our dPCR thermal protocol listed in Table 1 (0 second denature, 0 second annealing and extension) to the current dPCR protocol recommendation of 15 seconds denature, 30 seconds anneal/extend. We performed this test using the PIK3CA p.H1047R titration. Figure 4 shows consistent quantification performance for low MAF targets between the two thermal profiles.

Summary

Digital PCR (dPCR) enables rare target detection even among a high amount of background non-target presence. The Combinati Absolute Q dPCR platform consists of a familiar microtiter-plate formatted consumable and a fully integrated instrument. The platform offers a simplified one-step workflow identical to traditional quantitative PCR (qPCR). The Absolute Q was used with the Applied Biosystems TaqMan Liquid Biopsy dPCR Assays to perform rare target detection for 5 hot-spot cancer mutations. A sensitivity of 0.1% MAF was demonstrated for each assay showcasing the Absolute Q’s cross-assay robustness for clinically relevant rare target detection.

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