Application Notes: Absolute Quantification

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.

Using in-line process controls to evaluate sample processing workflow efficiency

Introduction

Environmental monitoring of wastewater samples for the presence of SARS-CoV-2 has demonstrated the potential to provide population level estimates of COVID-19 disease burden. 1 While routine monitoring of community wastewater samples for the presence of SARS-CoV-2 viral targets can be complimentary to clinical testing of individuals, quantifying dynamic changes in the number of viral RNA molecules can help identify trends in potential COVID-19 cases within the community. It’s important to recognize the variability arising from differences in sample preparation methods to better track the changes in viral target concentration over time. Process controls are widely used to address sources of variability. Spike in controls, such as Bovine Coronavirus, is one of the most commonly employed targets to provide insight into this variability in wastewater surveillance efforts.

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Digital PCR provides more accurate quantitative results

Since dPCR provides standard curve-free absolute quantification of targets, the resulting quantitative data is more accurate and more reproducible than typical qPCR methods. Multiplexing provides additional quantitative data that can be leveraged for normalization of downstream results and help limit the impact of upstream variability. The Combinati SARS-CoV-2 Wastewater Surveillance 4-plex assay was specifically designed to quantity 2 SARS-CoV-2 viral targets (N1 and N2) alongside a human fecal normalization control (PMMoV) and the sample process control (BCoV) in a single digital PCR reaction. This enables 4x the amount of data to be collected per sample run than many traditional qPCR reactions. Using four separate color channels enables simplified detection of each target without the need for multiple standard curves or tedious manual gating of multiple positive clusters per channel.
This technical note highlights how the sample process control Bovine Coronavirus (BCoV) can be utilized to provide information regarding the success and efficiency of nucleic acid extraction without impacting the sensitivity or accuracy of SARS-CoV-2 detection and quantification.

Using a process control to evaluate sample extraction efficiency

Input Considerations

A spike in process control is intended to provide quantitative information about sample loss during sample processing workflow. Typically, a process control is spiked into the initial sample before any processing takes place, and the presence of that control at the end of the workflow serves as an indicator of successful sample recovery.
For the SARS-CoV-2 Wastewater Surveillance 4-plex assay, the endpoint readout of the Bovine Coronavirus target (BCoV) is an absolute quantification of the total number of molecules, determined through digital PCR. By quantifying the control material both prior to spike in and after the processing workflow is complete, an independent measure of the overall yield of the workflow can be calculated. This is enables accurate evaluation of workflows without depending on endogenous targets that could naturally vary from sample to sample.

Interpreting Digital PCR Data

Results reported by the Absolute Q Analysis software are the concentration of each target in copies per microliter (cp/µL) in each dPCR reaction. To calculate the concentration of targets in the original input sample, first calculate the total number of molecules in the dPCR reaction by multiplying the concentration by the 9µL total volume of the dPCR reaction (Equation 1).
Next, to calculate the concentration of the sample used as input into the dPCR reaction, divide the quantity calculated using Equation 1 by the volume of sample used as input. For example, if 5µL of extracted RNA from wastewater was used per dPCR reaction, divide the quantity by 5µL to obtain the original extracted RNA concentration.

Evaluating workflow efficiency

Comparing the concentration of BCoV spiked into the original wastewater sample to the amount of BCoV recovered post processing can provide insight into the overall yield of the workflow. For specific suggestions on how to quantify and use BCoV as a wastewater sample process control consult the SARS-CoV-2 Wastewater Surveillance Kit Instructions for use.

Using Bovine Coronavirus as a process control for wastewater SARS-CoV-2 monitoring

Here we highlight the consistent quantification of BCoV using an example sample extraction workflow. After reconstituting the BCoV control material (materials and methods), 5 serial 10-fold dilutions were prepared, extracted and quantified using the SARS-CoV-2 Wastewater Surveillance Kit. In addition to the BCoV serial dilutions, a SARS-CoV-2 positive reference material and water-only conditions were included as positive and negative extraction controls.

All samples were tested using the SARS-CoV-2 Wastewater Surveillance 4-plex Kit. Figure 1 highlights the consistency of quantification between replicates as well as linearity of the serial dilutions of BCoV (p<0.001). The results suggest that the RNA extraction was successful and the quantification was consistent across the range of input material (Figure 1A). The positive and negative process controls as well as PCR control, which included controls for all 4 assay targets, behaved as expected (Figure 1B-D).

To verify the BCoV concentration obtained from the multiplex assay, a simplex BCoV assay was used to quantify the same purified RNA samples. The average concentration of each sample from the simplex assay showed high is completely in concordance with the concentration from the multiplex assay with a significant Pearson correlation (R2 = 1.0, P<0.0001) Table 1.

Calculating input concentration by dPCR

Using the dPCR data, concentration of the original BCoV material can be calculated using Equation 1. Since one microliter was used as input into each PCR reaction, concentration of the original material can be calculated by accounting for the initial dilution factor for sample extraction. Based on the results, the concentration of the original BCoV input material prior to extraction is 2.39E+06 (Table 2).

Accuracy and sensitivity for SARS-CoV-2 targets remain high

To demonstrate the sensitivity and accuracy of the 4-plex assay in the presence of spike in process control material, we validated the quantification accuracy of the 4-plex assay using three levels of SARS-CoV-2 target concentrations with backgrounds of 5-log concentration range of BCoV (materials and methods).
Three concentrations of SARS-CoV-2 reference RNA (stock , 10-fold and 100-fold dilutions) were added to each point of the BCoV extracted RNA dilution series along with a constant quantity of PMMoV ssDNA (materials and methods). Each mixed sample was then tested using the SARS-CoV-2 Wastewater Surveillance 4-plex assay. The 3 serial 10-fold SARS-CoV-2 reference RNA control materials were also tested without BCoV and PMMoV to evaluate the effects of the spike in material on quantification.
As shown in Figure 2, while the BCoV concentration decreases with each serial dilution point, the quantity of the N1 and N2 remain constant for the stock concentration (Figure 2a), 10-fold dilution (Figure 2b) and 100-fold dilution (Figure 2c) of SARS-CoV-2 reference material. The level of PMMoV (red-bars) remained constant for all conditions tested and yielded a concentration of 46.37 copies/µL (±4.78cp/µL) across all 47 reactions. The measured concentrations with and without BCoV spike-in RNA in the reactions are highly correlated for both N1 and N2 targets as shown in Figure 2d.

Workflow/Materials and Methods

Nucleic Acid Material

Extracted RNA, synthetic construct and standard reference materials were used in the experiments. The Bovine Coronavirus RNA has been extracted from the modified live virus BOVILIS CORONAVIRUS (Merck Animal Health). A 10-dose was reconstituted in 2 milliliters 1x TE buffer (Thermo Fisher Scientific, Waltham, MA) and serially diluted to 1:10, 1:100, 1:1,000, 1:10,000 and 1:100,000. RNA extraction was performed on Maxwell RSC 16 (Promega, Madison, WI). 50 µL of each dilution were extracted along with extraction controls including nuclease free water as negative control and positive control AccuPlex SARS-CoV-2 Positive Reference Material (SeraCare, Milford, MA). All samples were eluted in 50 µL nuclease free water and stored in -80ºC freezer. Additionally, single stranded synthetic DNA for Pepper Mild Mottle Virus (PMMoV) and Exact Diagnostic SARS-CoV-2 RNA control material (Bio-Rad, Hercules, CA) with three dilutions (stock, 1:10 and 1:100) were used in the digital PCR experiments.
Two hydrolysis probe-based assays were used in the study. 

Combinati SARS-CoV-2 Wastewater Surveillance 4-plex Kit

The Combinati 4-plex SARS-CoV-2 Wastewater Surveillance assay specifically detects SARS-CoV-2 N1 (FAM) and N2 (HEX) as well as the targets for BCoV process control (TAMRA) and PMMoV human fecal normalization control (TYE) in a single multiplexed reaction. A simplex assay with a probe in the TAMRA channel was used to measure the extracted BCoV RNA.
After preparing the one step RT-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. Figure 3 details the thermal cycling and reagent preparation protocols for RT-dPCR on the Absolute Q. Following the RT-dPCR, the sample concentrations were determined using the Absolute Q Analysis Software with the following threshold for each target FAM, 5000 fluorescence units, HEX 1000 fluorescence units, TAMRA 1300 fluorescence units). Statistical Analyses were performed using Graphpad Prism 9.1.0 (La Jolla, CA)

References

Wu, Fuqing, et al. “SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases.” MSystems, vol. 5, no. 4, 2020, doi:10.1128/msystems.00614-20.

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.

Digital PCR Wastewater Surveillance: Detect SARS-CoV-2 alongside human fecal and process controls

Background/Significance

Wastewater surveillance of SARS-CoV-2 has been shown to be a useful predictor of potential outbreaks. However, for meaningful interpretation of SARS-Cov2 data, both quantification accuracy and data normalization are critical. Moreover, reverse transcription qPCR (RT-qPCR) – the most commonly used method – reports a threshold cycle that requires a standard curve to provide quantitative information. RT-qPCR’s reliance on standard curves means the accuracy of the measurements depends directly on the accuracy and reproducibility of the reference materials used. These factors combined make interpretation of data on a broad scale extremely challenging. 

Digital PCR (dPCR) provides absolute quantification, and when combined with multi-colored multiplexing, can incorporate controls in a single reaction to provide normalized results for multiple targets. These results enable more accurate comparisons between samples with varying upstream sample preparation methodologies and more robust longitudinal monitoring.

Benefits of the Absolute Q for SARS-CoV-2 Wastewater Monitoring

  • Quantification of three wastewater-specific genomic targets in a single dPCR reaction
  • Integration of Human Fecal and Process Controls allows normalization and recovery efficiency to be calculated without additional reactions
  • Single instrument qPCR-like workflow in under 2 hours

The Combinati 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). In addition to these three targets, the assay also integrates the process control Bovine Coronavirus (BCoV). This inactivated virus, which is not generally present in community sewer systems, is spiked into the initial raw sewage sample before downstream processing. Its similarity to human SARS-CoV-2 allows it to be used as a surrogate to monitor the overall efficiency of sample processing.

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Assay Design

The Combinati SARS-CoV-2 Wastewater Surveillance 4-plex assay combines four sets of primers/probes into a single multiplexed assay. The assay is composed of two genetic targets N1 and N2 on the SARS-COV-2 N gene, a matrix recovery target (aka. process control) on Bovine coronavirus genome (BCoV), and a Human fecal normalization control target on Pepper Mild Mottle virus (PMMoV). N1 and N2 have been reported to be sensitive and specific for quantifying SARS-CoV-2 RNA in wastewater. Bovine coronavirus is an enveloped virus with a single stranded RNA genome similar to SARS-CoV-2, but not usually present in wastewater. The plant pathogen Pepper Mild Mottle Virus (PMMoV), an indicator of human fecal pollution, is widespread and abundant in wastewater from the United States.

N1, N2, BCoV and PMMoV targets were labeled with FAM, HEX, TAMRA, and TYE665, respectively (Figure 1). All primers were checked for target sequence specificity using NCBI Primer-BLAST1. Primers and probes were also evaluated for primer dimers and cross primer interactions using Multiple Primer Analyzer (Thermo Fisher Scientific, Waltham, MA).

Absolute Q Workflow and Experiment Materials

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 (Figure 1). The prepared MAP16 plate was then loaded on the Absolute Q. Figure 2 details the thermal cycling and reagent preparation protocols for RT-dPCR on the Absolute Q.

Figure 1. Workflow for the SARS-CoV-2 Wastewater Surveillance 4-plex assay

Figure 2 (right). Absolute Q digital PCR thermal parameters and reagent preparation table

Assay Performance: Sensitivity

Accurate quantification of SARS-CoV-2 RNA is critical when comparing results between locations or performing longitudinal surveillance. We prepared and quantified a serial dilution of the commercially available SARS-CoV-2 control material (Exact Diagnostics, SKU: COV019), which contains both the N1 and N2 targets to demonstrate the quantification accuracy of the assay. 

The serial dilutions consisted of three 4-fold dilutions to simulate a range of viral RNA concentrations. These RNA dilutions were spiked into a constant background of BCoV and PMMoV control materials at approximately 500 and 1000 copies per reaction respectively. Figure 3 summarizes the results of this N gene assay sensitivity dilution series.

Figure 3. Results from serial 4-fold dilutions of Exact Diagnostic SARS-CoV-2 control material into a background of BCoV and PMMoV control material at approximately 500 and 1000 copies per reaction respectively. A) The X-axis represents the targeted copies/reaction and Y-axis represents the observed concentrations of the N gene targets, N1 (purple) and N2 (orange). Two-dimensional partition scatters for the N1 (FAM) and N2 (HEX) targets across the RNA control material dilution using 2µL each of the (B) stock control material, (C) 4X dilution (D) 16X dilution and (E) 64X dilution.

The targeted concentrations of the N1 and N2 genes across the dilution series were 550, 137.5, 34.4 and 8.5 copies per reaction with the observed concentrations reported in Table 1.  The concentrations are significantly correlated for both N1 and N2 with Pearson R2 values of 1.0 (p<0.001). Across each SARS-CoV-2 dilution point, both BCoV and PMMoV remained constant at 471.8 (±32.4) and 1194.5 (±77.3) copies per reaction respectively.

Table 1. Average observed N1 and N2 copies per reaction using the SARS-CoV-2 Wastewater Surveillance 4-plex assay. At least 3 replicates were run for each condition. In addition, a water control was included. In one of three NTCs, a single false positive partition was identified for N1 and N2.

Assay Performance – Specificity

To demonstrate the high specificity and low cross reactivity of the 4-plex assay, individual materials and mixtures of the target control materials were tested against the 4-plex assay. The 4-target PCR control material demonstrated successful amplification in all four targets (Figure 4a) and the no-template added negative control showed zero false positive amplification events (Figure 4b). Subsequent tests of individual target control materials demonstrated high specificity for the intended target for each assay component (Figure 4c-f).

Figure 4.  Partition amplification plots shown for each target N1 (blue channel, FAM), N2 (green channel, HEX), BCoV (yellow channel, TAMRA) and PMMoV (dark red channel, TYE665) by rows are: A) a PCR control 4-plex containing single stranded DNA (ssDNA) N1, N2, and PMMoV controls alongside inactivated BCoV RNA control material; B) water only no template control which yielded no false positives; C) N1 ssDNA control; D) N2 ssDNA control; E) PMMoV ssDNA control;  F) BCoV RNA control.

Using Human Fecal and Process Controls for Data Comparisons

Viral load present in wastewater can be impacted by a variety of factors, including differences in preparation methods as well as the total amount of human fecal matter present. Understanding the amount of human fecal matter relative to the quantitative measurement of SARS-CoV-2 enables more accurate data interpretation for community level testing. The SARS-CoV-2 Wastewater Surveillance 4-plex assay incorporates two orthogonal controls in order to help interpret results. Quantification of those controls (BCoV, process control and human fecal control PMMoV) in the same reaction as the SARS-CoV-2 N gene targets enables more precise comparison between samples. The following dataset illustrates one option for how the BCoV and PMMoC controls can be used to interpret SARS-CoV-2 wastewater-based epidemiology data.

Figure 5. Concentration of N2 (orange) and N1 (purple) SARS-CoV-2 targets in contrived samples. Using the SARS-CoV-2 Wastewater Surveillance 4-plex assay, three replicates were tested per contrived sample.

We tested four contrived samples using the SARS-CoV-2 Wastewater Surveillance 4-plex assay. All four samples demonstrated similar overall N1 and N2 quantities (Figure 5).  However, in the same four samples, the measured PMMoV concentration varies significantly (Figure 6a).

Figure 6. A) Initial concentration in copies per reaction of each contrived sample for the N1 (purple), N2 (orange) and PMMoV (red) targets. B) Chart reflects the concentration of N1 (purple) and N2 (orange) normalized to the median PMMoV value of 5357 copies of PMMoV.  Using the SARS-CoV-2 Wastewater Surveillance 4-plex assay, three replicates were tested per contrived sample.

In order to make viral load comparisons, normalization to the human fecal marker PMMoV can be used to account for information such as the size of the community sampled. In this example, the ratio of N1 or N2 quantities to the concentration of PMMoV was normalized to the median PMMoV concentration measured in this dataset (5357 PMMoV copies) as detailed in the Methods section. While Samples B and C demonstrated very similar levels of N1 and N2, their PMMoV concentrations varied substantially (Figure 6a). After normalization to the PMMoV median, Sample B had the highest relative abundance of SARS-CoV-2 targets (Figure 6b).  

Finally, the process control (BCoV) levels can be used to verify there are no large discrepancies in sample preparation efficiencies between the samples. As shown in Figure 7 the measured BCoV concentrations are comparable to one another across the four samples. Assuming an equivalent amount of control material was spiked into the native sample at the start of processing, this would indicate processing variations were minimal.

Figure 7. Concentration of BCoV across contrived samples using the SARS-CoV-2 Wastewater Surveillance 4-plex assay. Three replicates were tested per sample.

Summary

When comparing quantitative data, consistent measurement techniques that introduce as few variables as possible are essential. Digital PCR (dPCR), which provides absolute quantification of targets without standard curves, enables the quantification of all targets (including process and internal controls) to produce more accurate and more broadly comparable wastewater datasets – even when upstream preparation methods vary. 

The Combinati SARS-CoV-2 Wastewater Surveillance 4-plex assay was designed to detect and quantify SARS-CoV-2 viral targets while simultaneously providing normalization and recovery data in a single reaction. Using both human fecal markers and an orthogonal process control, sources of variability such as fecal load variation due to population levels or inconsistencies in sample processing can be accounted for. With high specificity, sensitivity, and best in class sample utilization of 95%, the Absolute Q provides more accurate and consistent quantification of these wastewater relevant targets.

Materials and Methods

Control Materials

Wet-lab validation of the assay has been performed using control materials. For assay specificity evaluation, single stranded DNA controls were used for the N1, N2 and PMMoV targets and Bovilis Coronavirus Calf Vaccine was used as the BCoV positive control. For assay sensitivity evaluation, the Exact Diagnostic SARS-CoV-2 RNA control material was used. For contrived samples, the N1, N2, BCoV, and PMMoV controls were mixed to create varying abundance ratios.

Normalization

To normalize the concentration of N1 and N2 with respect to PMMoV for the contrived sample experiment, the following steps were performed. First, the concentration of each target (N1, N2, and PMMoV) were multiplied by the reaction volume to calculate the total copies per reaction. Subsequently, the concentration of N1 or N2 was divided by the concentration of PMMoV to obtain a ratio. Finally, the ratio was multiplied by the median PMMoV concentration for the dataset.

References

  1. “Primer Designing Tool.” National Center for Biotechnology Information, U.S. National Library of Medicine, www.ncbi.nlm.nih.gov/tools/primer-blast/.

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.

Establishing the Limit of Detection for the |Q| SARS-CoV-2 Triplex Assay

Background

Widespread testing has been proven to be an important tactic to combat widespread infections during the COVID-19 pandemic. Many types of tests have been brought to the market in an effort to expand test availability to all corners of the globe. However, in order to choose the most appropriate option, it is important to understand and consider both the sensitivity and accuracy of the test in addition to its availability. False negatives could lead to an increase in community spread and significantly increase the risk of large scale outbreaks.

Limit of detection (LoD), also known as analytical sensitivity, is often used to describe the lowest concentration of input that can be reliably distinguished from a blank. In this study, we characterize the LoD of the Combinati |Q| SARS-CoV-2 RT-dPCR Triplex Kit by diluting reference materials into a pooled negative matrix to determine the lowest concentration at which the assay can reliably identify the sample as containing SARS-CoV-2 targets.

The traditional quantitative PCR (qPCR) approach, the current gold standard for COVID-19 diagnosis, generates results in terms of Ct or Cq. These values do not provide quantitative measurements of the virus without a standard curve. Instead, a predetermined qPCR threshold result determines if a sample is deemed positive or negative. In contrast to this binary result provided by qPCR, digital PCR (dPCR) provides absolute quantification of nucleic acid targets without a standard curve. Each dPCR assay provides a quantitative measure of the targets present in the original sample. In this study, we present quantitative dPCR data to demonstrate the ability to use the Combinati |Q| SARS-CoV-2 RT-dPCR Triplex Kit for applications that look to quantify viral load changes between samples or over time.

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Experimental Protocol

Serial dilution of input materials: Synthetic virus from SeraCare (AccuPlex™ SARS-CoV-2 Reference Material Kit, Cat No. 0505-0126) was serially diluted into a pooled negative swab matrix (VTM/UTM). The samples contained target concentrations from 1600cp/mL to 50cp/mL in 2-fold dilutions.

Nucleic acid purification: The Promega Maxwell RSC16 (Cat No. AS4500) and the Maxwell® RSC Viral Total Nucleic Acid Purification Kit (AS1330) were used to extract RNA from the dilution series samples. For each extraction, 150µL of sample input volume and 60µL elution volume were used.

Digital PCR protocol: For each dPCR run, 6.5µL of extracted sample was combined with 2.5µL of RT-dPCR MasterMix and 1µL of the Triplex Assay. 9µL of the reaction mixture was then loaded into a single well of the MAP16 consumable. Each MAP16 plate run included one NTC (no template control) to ensure that no contamination occurred during testing.

Thermal cycling protocol for the Absolute Q Digital PCR Platform is shown below in Table 1.

Table 1 Thermocycling Parameters

Interpretation of assay results: The determination of whether a sample is “positive” for SARS-CoV-2 was made according to Table 2.

Table 2 Interpretation of Assay Results

As described in the table, any sample that contained two or more positive partitions for either N1 or N2 target is considered positive for SARS-CoV-2.

Limit of detection determination and confirmation: Two sets of experiments were performed to determine the LoD. First, nine replicates for each of the dilutions from 1600cp/mL to 50cp/mL were tested to determine the preliminary LoD. Subsequently, the three lowest concentrations that demonstrated positive signal for all nine replicates (100% accuracy) were selected for additional testing. For each of these concentrations, 20 extraction replicates were tested to confirm the limit of detection.

Results

Preliminary LoD determination: Results from the LoD determination experiment are summarized below in Table 3.

Table 3 Preliminary LoD Determination Results

For all concentrations down to 200cp/mL, nine out of nine replicates (100%) resulted in positive calls for both N1 and N2 targets. For 100cp/mL input, only four out nine replicates were called correctly for N1 and six out of nine replicates were called correctly for N2. Based on these results, the three concentrations selected for the confirmation experiment were 800cp/mL, 400cp/mL, and 200cp/mL.

LoD confirmation: For LoD confirmation, 20 extraction replicates were performed for each of the three concentrations selected. The LoD is defined as the lowest input concentration that results in greater than or equal to 95% of all true positive replicates testing positive for SARS-CoV-2. Results for the confirmation experiment are summarized below in Table 4.

Table 4 Confirmation of LoD

The LoD was determined to be 200 cp/mL, as 20 out of 20 replicates were correctly identified as positive for SARS-CoV-2 for both N1 and N2.

Quantitative measurement of serially diluted samples: As dPCR provides absolute measurements instead of a cycle number, small changes can be accurately detected and quantified. Figure 1 shows the number of positive partitions for each of the dilutions used in the preliminary LOD study.

Figure 1. Number of Positive Partitions with Various Input Concentrations

A linear relationship between the input concentration and number of positive partitions detected was identified (N1 R2 = 0.994, N2 R2 = 0.993). This provides strong evidence for the feasibility of accurate and precise quantitative monitoring of viral presence changes using the Absolute Q Digital PCR Platform.

Results

In this study, we established the limit of detection of the Combinati |Q| SARS-CoV-2 Triplex Kit as 200 cp/mL and defined the protocol used to determine the LoD. Additionally, we demonstrated the ability to accurately quantify across a large range of input sample. In summary, the Combinati |Q| SARS-CoV-2 RT-dPCR Triplex kit combined with the Absolute Q Digital PCR Platform enabled highly sensitive detection and quantification of SARS-CoV-2 when coupled with the Promega RSC for nucleic acid purification. The quantitative measurement provided by the assay can be used for a wide range of applications, including tracking viral load changes and wastewater monitoring.

Using Digital PCR for Optimization of SARS-CoV-2 RNA Extraction Protocol

Background

RNA extraction is a critical step in COVID-19 molecular testing. Loss of viral RNA during the extraction step can result in false negatives. Therefore, optimization of the RNA extraction protocol to ensure consistent and high yield recovery of viral RNA could potentially improve COVID-19 testing accuracy. The goal of this study is to demonstrate how digital PCR can be used to optimize conditions for viral RNA extraction using verified molecular controls. Digital PCR may also be used as a quality control tool to monitor sample preparation consistency across facilities and labs.

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Evaluation of CRISPR-Cas9 Mediated Genome Edits with the Absolute Q Digital PCR platform

Background

Genome editing through the use of the CRISPR/Cas9 system has become a tool which is widely used across many scientific disciplines. As more labs employ the use of CRISPR/Cas 9 to introduce customized changes to targets, it is equally critical to have a method of monitoring the success and efficiency of these gene editing processes.

Digital PCR, which provides higher precision quantification of nucleic acids, is aptly suited for the analysis of genome editing applications such as CRISPR/Cas9 mediated knock-ins and knock-outs. This is largely enabled by dPCR’s fundamental principle of absolute quantification, which provides quantification of nucleic acid targets without the need for orthogonal standard curves. This method of quantification is more consistent and more accurate, particularly for rare or low concentration targets. This application note highlights the Absolute Q digital PCR platform paired with a 2-probe assay design designed in collaboration with Integrated DNA Technologies (IDT) which was used to detect and quantify both CRISPR meditated knock in and knock outs with precision and accuracy. 

The Absolute Q is a vertically integrated digital PCR platform which enables a simplified digital PCR workflow – using a single instrument and a single consumable to deliver complete results in under 2 hours. The microfluidic array partitioning (MAP) plate provides routine and consistent generation of 20,000 identically sized partitions, dispersing over 95 percent of the sample across each dPCR reaction every time. Unlike many available digital PCR systems, the workflow is identical to qPCR. This means every user can generate consistent and accurate digital PCR every time with minimal hands-on steps. 

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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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