Uniformity Across the Block  

***This is a Premium Content Article that needs to be unlocked for tables and graphs to be visible***

With its unprecedented throughput, scalability and speed, next-generation sequencing (NGS) enables researchers to study biological systems at a level never before possible. Today’s complex genomic research questions demand a depth of information beyond the capacity of traditional DNA sequencing technologies. Next-generation sequencing has filled that gap and become an everyday research tool to address these questions across translational research areas such as clinical diagnostics, agrigenomics, and forensic science.

However, there is a challenge. One failed NGS run is a huge cost on the laboratory – in fact, they can sometimes cost as much as $3,000.

In order to make sure that sequencing is cost-efficient, as well as streamlined and accurate, laboratories need to be confident that imprecise, low uniformity qPCR techniques are not ruining their runs. This can have a lot to do with the instrumentation used. A multitude of factors within each PCR system, from temperature control to light bleed-through, can impact on how dependable each run is. By delivering complete uniformity, manufacturers can guarantee that each run is completed reliably and accurately.
Innovative new technologies are becoming available in commercial instruments to ensure this. In this article we will look how instruments such as the Eco 48 real time PCR system (PCRmax) deliver exceptional uniformity across the block, and the impact this level of reliability can have within a working laboratory.

Improving the Performance of NGS Technology

Precise DNA amplification is a fundamental pursuit in genomics research. Concomitantly, the development of high throughput NGS technology is often considered one of the most transformative innovations in biological sciences of the past 30 years. Today, this powerful technology has superseded traditional PCR techniques such as capillary and gel based Sanger sequencing as the industry standard.
NGS technology enables researchers to simultaneously process millions of parallel DNA sequences many orders of magnitude faster than traditional sequencing. However, NGS remains a high cost process; the price of a commercially available NGS system can range from around $150,000 US to well in excess of $1,000,000, and the cost of driving these processes is equally high. Optimising the conditions of each run to minimise the risk of failure and maximise return on investment is therefore a critical priority during method development. Target library quantitation has been identified as an area where cost effective technologies are available to streamline this NGS workflow. 
The NGS process requires the preparation of libraries in which fragments of target molecules are fused with adapters, followed by amplification and sequencing with a polymerase chain reaction (PCR). The size of the target DNA fragments in the final library is a critical parameter for NGS library construction. If too little template is loaded onto the NGS platform then the run will have low efficiencies. If too much is loaded onto the platform then the run not only risks low efficiency but increased probability of complete failure. Robust and reliable library quantification is therefore critical. 
Quantitative PCR (qPCR) is the technique of choice for NGS library quantitation. However, not all PCR systems deliver sufficient thermal uniformity to perform with the high repeatability and accuracy required for secure NGS system loading. Moreover lengthy qPCR protocols severely impact the efficiency of the entire NGS run. With the price of high performance qPCR instruments being outweighed by the cost of a single failed run, employing a robust quantitative qPCR technique is considered one of the simplest and most cost-effective ways to achieve consistent NGS. 
Choosing the Right PCR Technique qPCR monitors the emissions from fluorescent primers during the annealing process to monitor strand amplification. Fluorescence signals are captured in real time to enable quantitative analysis at the appropriate phase of the reaction. This overcomes the imprecision associated with traditional, semi-quantitative end-point detection, including unaccounted variation in PCR efficiency between samples and errors when extrapolating back to the starting quantity.

Temperature control is at the heart of the qPCR process; it dictates whether primers will bind efficiently and whether the polymerase enzymes will work optimally. To achieve high accuracy, a real time PCR thermal system must maintain a uniform temperature across the entire heat block, ensuring that all samples in the well proceed through the reaction at an equal rate. However, standard qPCR systems only have a thermal accuracy of around ±0.5ºC at the 50 to 60ºC range. This is often insufficient to precisely define the cluster density, and an NGS run may risk failure if the qPCR platform under or over represents the true library concentration. The importance of precision is highlighted by stringent MIQE (Minimum Information for Publication for Quantitative Real-Time PCR Experiments) guidelines for qPCR experiments, which demands increasingly sensitive qPCR technology. 
Traditional qPCR systems employ Peltier-heated thermal blocks to drive thermal cycling. Heating the whole surface of these solid blocks is an energy intensive process. Most Peltier based systems are designed to exceed the required reaction temperature before equilibrating to a desired plateau. The time it takes for all wells within the block to reach this point results in long run times. More significantly, this method of heating contributes to high thermal non-uniformity (TNU) values of ±0.5ºC and poor thermal ramp-rates.
A combination of imprecision and inefficiency, traditional solid-block qPCR is poorly suited to high performance applications.

Recent advances in qPCR heating technology overcome these issues. Modern systems use a precisely uniformed hollow silver block through which a conductive fluid is passed. A single Peltier device is used to heat and cool the fluid which is then circulated evenly across all the sample wells by opposing agitators. This ensures robust thermal performance, with TNU values below ±0.1ºC at 95ºC, and reduced run times. A standard 40 cycle PCR protocol that uses this system takes about 40 minutes to complete, while an optimised process can take only 15 minutes. 
Advances in fluorescence monitoring technology have also improved the performance of qPCR techniques. High-performance optical systems enable real-time detection of up to four targets in a single reaction and advanced detector arrays monitor the fluorescence from all wells, allowing the system to record every well, filter and cycle without missing a single data point. Finally the use of stable light emitting diodes (LEDs) contributes to accurate data generation and increases instrument longevity, decreasing depreciation costs. 
The following case study illustrates how improvements in the thermal uniformity across the block deliver the sensitivity, precision and overall efficiency required for high performance NGS applications.  
Case study: Improving the Precision of qPCR 48 replicate samples were subjected to a High Resolution Melt (HRM) protocol using the PCRmax Eco 48. HRM enables precise analysis of genetic variations, such as quantifying single nucleotide polymorphs (SNPs), and is one of the most thermally demanding protocols in terms of accuracy.

Each of the 48 sample wells were filled with 1x108 copies of the starting template (100bp template based on Lambda phage DNA) in a 10µl final volume. The plate was sealed and centrifuged for 1 minute at 12000 rpm and the un-optimised 40 cycle PCR protocol was performed in 43 minutes total. The template was amplified for 40 cycles (95°C, 10s; 60°C, 30s) using the GoTaq® qPCR Master Mix (2x) from Promega (part code A6001). Fluorescence data was collected at the end of the 60°C step. The results were analysed using Eco study software to determine the quantitation cycle (Cq), the point at which fluorescence can be detected, and the melting temperature (Tm) values for each of the 48 replicates.
Figure 2 shows the baseline corrected amplification plot for all 48 wells. The graph clearly demonstrates precision of amplification across the entire plate. Analysis of the data showed an average Cq of 13.31 with a standard deviation of ±0.061. This equates to a coefficient of variation (%CV) across the plate of just 0.46%, indicating exemplary precision. 
Tm is determined by running a melting stage following the amplification of the PCR product and is one of the most effective measures of block uniformity. The amplified product melted in the 75ºC to 95ºC range and the Eco 48 measured the fluorescence with every 0.1ºC temperature change, the accuracy required to detect class IV SNPs with greater than 99% accuracy. Figure 3 shows the normalised melt curve. 
Tm average across all 48 well plates was recorded as 84.45ºC with a standard deviation of ±0.058, equating to a %CV across the plate of just 0.07%. This suggests that excellent temperature uniformity is achieved across the block. Figure 4 summarises the results for this experiment. 

Driving the Future of Genomics
Systems that employ precise thermal accuracy improve the analytical productivity of all PCR applications. However, it is the increased confidence that greater thermal control provides users during high cost NGS processes that have made high performance qPCR an essential feature of routine sequencing. qPCR instruments with innovative block technology, such as the PCRmax Eco 48, deliver complete heating uniformity and rapid cycles in under 40 minutes, with the sensitivity to deliver ±0.1ºC uniformity across the whole block instantly after every temperature change. These leaps in efficiency allow users to achieve consistent NGS runs, helping to further their critical genomics research.