Application Notes: Medium Multiplexity

Robust Quantification of Next Generation Sequencing Libraries using Absolute Q Digital PCR

Highlights

  • Consistent reagent partitioning in to greater than 99% of expected partitions compared to 60.9% on emulsion dPCR platform
  • Improved separation of positive and negative partitions in 2-dimensional threshold view
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Introduction

Advances in next-generation sequencing (NGS) technologies had accelerated the discovery of actionable genomic targets. Accurate quantification of the final library products is critical to maximizing both data quality and output. However, conventional quantitative PCR (qPCR) methods commonly used to assess NGS library concentrations do not evaluate the concentration of complete library fragments. Moreover, in some cases, the amount of final library product is limited, and the input requirement for consistent qPCR quantification can become a hindrance. 

Multiple studies have demonstrated that utilizing digital PCR (dPCR) as a quantification tool for NGS libraries before sequencing helps optimize sequencing run performance, data generation, and data quality1,2. Digital PCR, which uses absolute rather than relative quantification, has superior accuracy and precision at low concentrations relative to qPCR. By leveraging multiplexed digital PCR reactions, users can identify and quantify specific library fragments representing sequenceable molecules (Fig 1).

In this technical note, the Absolute Q Digital PCR Platform was used to perform NGS library quantification of sequenceable library molecules. Four NGS libraries with various insert sequence lengths were quantified in this study.

diagram of NGS library quantification by dPCR

Materials and Methods

Library preparation, separation and dilution

A single mixed-size ATAC-seq library was separated based on fragment length (BluePippin) to create four separate NGS libraries with average fragment lengths of 300, 500, 700, and 1000 base pairs. After size separation, each library was amplified using Q5 polymerase (NEB) and cleaned up using SPRIselect (Beckman Coulter). The amplified libraries were then diluted at 1:200,000 to create the “Dilution 1” then subsequently diluted serially 1:4 for a total of 6 dilutions for each of the 4 fragments lengths

Library preparation, separation and dilution

Digital PCR quantification of the NGS libraries was performed using 5µL of the diluted NGS library per reaction. A detailed breakdown of the digital PCR reagents is listed in Table 1. After preparing the dPCR mix, 9µL of the reaction mixture was loaded into a well of the MAP16 plate, followed by an overlay of 15µL of isolation buffer. The prepared MAP16 plate was then loaded on the Absolute Q. The following thermal parameters were used for each digital PCR run: 96°C hold for 10 minutes, 45 cycles of 95°C denaturation for 5 seconds, 61°C annealing and extension for 30 seconds. Data were collected using the FAM and HEX optical channels.

NGS library quantification reagent preparation

Analysis on Absolute Q

Combinati Absolute Q Analysis Software was used to calculate the concentration of the NGS libraries for each dilution series. The software reports the concentrations of targets that are FAM positive only, HEX-positive only, and FAM/HEX double-positive. By design, partitions containing complete and sequenceable library fragments are positive for both the P5 (FAM) and P7 (HEX) probes. The double positives are represented as a cluster in the upper right quadrant (green dots) of the two-dimensional fluorescence scatter view (Figure 1b). The concentration of double positives was then used in the following equations to determine the concentrations of the original library stock for each dilution point:

Results

Quantification of sequenceable library fragments

The four libraries, which vary in fragment length, were each quantified on the Absolute Q across six serial 4-fold points. The reported concentration in copies/µL from the Absolute Q for the dilution series for each library with specific fragment size are shown in Figure 2. As expected, the observed concentration decreased by approximately 4-fold between each point of the dilution series across all conditions. Using the reported concentration and Equations A and B, the original concentration of each NGS library were calculated to be 2.65nM (±0.78), 3.06 nM (±0.74), 3.06nM (±0.74), 4.63nM (±1.22) for the 300, 500, 700, and 1000 bp NGS libraries respectively (Table 2).

Advantages of microfluidic array partitioning for consistent reagent partitioning

An additional benefit of using Microfluidic Array Partitioning (MAP) technology to perform dPCR is the robustness of reagent partitioning. MAP technology leverages a micro-molded plastic dPCR plate with fixed volume arrays and ensures partitioning across >95% of the available partitions is achieved for all dPCR reactions.

To compare the partitioning efficiency between MAP and emulsion-dPCR, the four NGS libraries were quantified using an emulsion-based dPCR platform in parallel with the Absolute Q. To compare the performance and consistency of reagent partitioning between the Absolute Q and an emulsion-based dPCR platform, the accepted partition or droplet count was compared across 46 paired dPCR reactions. These NGS library quantification reactions were run in parallel on each platform using the same assay and library template material.

Out of a total of 20,480 fixed partitions per MAP dPCR reaction array, the mean accepted partition count for the Absolute Q reactions was 20,412 (±127 partitions). Out of the 20,000 droplets expected per reaction, the mean accepted droplet count for the emulsion-based dPCR reactions was 12,138 (±1267 droplets). For this dataset, the minimum number of accepted partitions for Absolute Q reactions was 19,645 and the minimum number of accepted droplets for the emulsion-based dPCR reactions was 8,629 (Figure 3).

Finally, a comparison of the representative 2-dimensional partition fluorescence plots between the emulsion-based dPCR platform (Figure 3b) and Absolute Q dPCR (Figure 3c) highlights the improved signal separation between the resulting double-positive partitions and negative partitions. The improved separation aids in consistent thresholding of dPCR data and as a result more robust and reproducible quantification results.

Summary

Quantification of NGS libraries by digital PCR is advantageous because it is possible to distinguish complete sequenceable library molecules and perform absolute quantification. Given that the final concentration of NGS libraries can vary based on the preparation method and performance of the method used, it is critical for each digital PCR reaction to be as consistent as possible to maintain robust quantification across a wide range of input concentrations.
The highest level of precision for dPCR quantification occurs when an average of 1.59 copies of target per partition is present in the reaction volume3. This means for high concentration samples the total number of accepted partitions is critical because more analyzed partitions can improve precision by bringing the average closer to 1.59. In the case of NGS library quantification, because the sample consists of amplified nucleic acids, the target concentrations are usually very high. The consistently high analyzed partition numbers of the Absolute Q ensure high levels of precision even at high target concentrations, ensuring high-quality NGS library quantification.
Here we demonstrated the capabilities of the Absolute Q to perform robust NGS library quantification across a wide range of fragment sizes and concentrations while achieving an exceptionally high number of accepted partitions per dPCR reaction.

References

  1. Robin, Jérôme D., et al. “Comparison of DNA Quantification Methods for Next Generation Sequencing.” Scientific Reports, vol. 6, no. 1, 2016, doi:10.1038/srep24067.
  2. White, Richard A, et al. “Digital PCR Provides Sensitive and Absolute Calibration for High Throughput Sequencing.” BMC Genomics, vol. 10, no. 1, 2009, doi:10.1186/1471-2164-10-116.
  3. Majumdar, Nivedita, et al. “Digital PCR Modeling for Maximal Sensitivity, Dynamic Range and Measurement Precision.” PLOS ONE, vol. 10, no. 3, 2015, doi:10.1371/journal.pone.0118833.

Improve sensitivity for rare targets by increasing total analyzed sample using digital pooling of digital PCR data

Introduction

A primary application of digital PCR (dPCR) is rare target detection. Absolute quantification, rather than cycle threshold values enable users to obtain the count of original target molecules in a sample – eliminating the need for interpreting amplification curves or relying on reference material to provide quantitative measurements. This improves both accuracy and reproducibility of concentration measurements of rare targets.
However, the limit of detection for any digital PCR reaction is dependent on several factors. These include the concentration of the target of interest and the volume of sample that can be loaded per reaction (Figure 1). In scenarios where the target of interest is present at a very low concentration, it may be necessary to run more than one reaction in order to sample enough volume to detect the target.

One example of rare target detection is the measurement of circulating tumor DNA (ctDNA) among normal circulating cell free DNA molecules (ccfDNA) from liquid biopsy samples of individuals diagnosed with cancer. In this application, the total number of ctDNA molecules can vary from patient to patient as well as from day to day; and are often very low in concentration – typically only several molecules per microliter. If the concentration of ctDNA molecules is exceptionally low, the amount of sample tested will directly impact the number of ctDNA molecules that can be detected. Furthermore, many existing digital PCR platforms suffer from lack of consistency and reproducibility in the total number of partitions generated due to limitations in the underlying partitioning technology, resulting in unreliable detection. This inconsistency can cause variability in the total amount of sample analyzed per reaction as well as between experiments. The wasted sample can contribute to sub-sampling error. Unlike emulsion based dPCR platforms, the microfluidic array partitioning (MAP) technology used in Absolute Q dPCR partitioning is entirely automated and highly consistent – utilizing an industry leading 95% of loaded sample volume across over 20,000 partitions each time.
This technical note highlights the digital pooling feature of the Combinati Absolute Q by leveraging an Applied Biosystems® TaqMan™ Liquid Biopsy Assay for the cancer mutation PIK3CA p.H1047R (cat. A44177). A contrived sample with mutant allele fraction at 0.1% was created and tested and digitally pooled across 4 dPCR reaction arrays (~81,920 partitions) .

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Workflow and Methods

DNA Mixture and dPCR reaction preparation

Using genomic DNA (Promega Human Male Control) and plasmid DNA containing the PIK3CA p.H1047R mutation, a DNA mixture of 0.1% MAF was prepared with final human genomic DNA concentration of approximately 1.15 ng/µL. The Applied Biosystems TaqMan Liquid Biopsy assay for PIK3CAp.H1047R which detects both the wild type (VIC) and mutation (FAM) alleles was used in this study. The digital PCR reactions were prepared according to the volumes indicated in Table 1.

Digital PCR reagent table

Reagent Preparation and Digital Pooling


After preparing the dPCR mix, 9µL of the reaction mixture was loaded into the MAP16 plate followed by an overlay of 15µL of isolation buffer. The prepared MAP16 plate was then loaded on the Absolute Q. Standard thermal parameters for use of the Applied Biosystems® TaqMan™ Liquid Biospy Assays on Absolute Q were used. Following the dPCR run, target concentrations were determined using the Absolute Q Analysis Software. A total of eight reactions were run using the samples and subsequently four arrays each were digitally pooled for a total of two replicates.
As an orthogonal test, a positive control at higher concentration of 0.1% MAF DNA mixture was prepared using the same plasmid and human genomic DNA. This DNA sample was used for 2 independent replicates and loaded at a final concentration of 33ng per reaction or approximately 10,000 wild type molecules and 10 mutation molecules per reaction.

Results

After dPCR was complete, all reactions were analyzed using the Absolute Q Analysis software. The dPCR results for the pooled low concentration 0.1% MAF sample across 4 arrays and high positive concentration control are shown in Table 2. Both conditions reported concentrations of the mutation and wild type PIK3CA molecules similar to the expectations. As indicated in Table 2, across approximately 81,545 digitally pooled partitions, the PIK3CA mutation allele was calculated to be at a concentration of 11.5 copies per reaction (cp/reaction) – similar to the positive control reaction which was determined to be 6.9 cp/reaction. Finally, the observed MAF of the digitally pooled low concentration sample was 0.1% MAF as expected.

Summary

Digital pooling is an effective method to increase sensitivity by increasing the amount of volume that can be analyzed for a given sample. In this technical note, four arrays (36µL dPCR Mix) of the MAP16 plate were used to analyze a total input sample volume of 28.8µL of a 0.1% MAF DNA mixture. Overall this method has the potential to be applied to a multitude of rare target detection applications in the precision medicine space such as monitoring treatment response, screening for minimal residual disease or rapid identification of mutations linked drug resistance.

Emulsion-free Digital PCR Measurement of Wastewater Related Targets using the SARS-CoV-2 Wastewater Surveillance 4-plex Assay

Background

Wastewater based epidemiology (WBE) enables tracking of biomarkers for specific pathogens to monitor for disease outbreak and spread. WBE’s utility in disease surveillance has been proven to be effective in monitoring for rare cases of disease.(1) To effectively monitor and quantity of SARS-CoV-2 viral targets from wastewater samples, it is critical to maximize the amount of information per testing reaction, minimize reagent waste, and control for external factors such as population size and sample processing efficiency.

Measuring wastewater related targets alongside robust controls can provide tangible metrics for normalization of results and help limit the impact of sample preparation variability. The SARS-CoV-2 Wastewater Surveillance 4-plex Assay was designed to detect the N1 and N2 SARS-CoV-2 viral RNA targets alongside the human fecal control, pepper mild mottle virus (PMMoV), and a spike-in sample process control, bovine coronavirus (BCoV).

In this study, we demonstrate SARS-CoV-2 detection and quantification alongside the normalization controls for four wastewater samples collected by the University of Arizona WEST center during their wastewater epidemiology testing efforts.

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  1. Asghar H, et al., Environmental surveillance for polioviruses in the global polio eradication initiative. J Infect Dis. 2014;210:S294–S303.

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 resulting in inconclusive SARS-CoV-2 results 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 inconclusive SARS-CoV-2 results 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

Reverse Transcription Digital PCR (RT-dPCR) enables higher accuracy quantification 

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 detection of SARS-CoV-2 viral RNA and subsequent diagnosis of COVID-19. However in a recent study, it was demonstrated that digital PCR (dPCR) provided better sensitivity for rare viral targets and in turn identifying patients who ultimately were diagnosed with COVID-19.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 SARS-CoV-2 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.

Click below to view or download a PDF of the assay protocol. 

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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, the reaction mixture was loaded into the MAP16 plate followed by an overlay 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 

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

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

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

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