The Road to Improved Next-Generation Sequencing Through Innovative PCR Instrumentation

Next-generation sequencing (NGS), one of the most significant technological advances in the the last 30 years, allows millions of DNA strands to be sequenced in parallel. This yields greater throughput and ensures that several genomes can be sequenced simultaneously.

NGS has become an everyday research tool in translational research areas such as clinical diagnostics and medical research. Its speed and sensitivity make NGS an attractive option to other sequencing modalities. A single failed NGS run, however, can cost as much as $3000.

Traditional qPCR systems suffered from imprecise temperature control, uniformity and light bleed-through, issues that compromise reliability. In recent years, innovative technologies have enabled tremendous progress in speed, read length and throughput. A most recent development, the Eco 48 real-time PCR system from PCRmax (a Bibby Scientific Company, Staffordshire, U.K.), can reliably provide uniformity across the block, thus assuring accurate NGS data.

NGS for the development of oncology drugs

NGS has become essential for assay development in disease detection and diagnosis, and in drug development. The genetic information obtained through NGS is used to better understand the process of disease development and how patient-specific characteristics influence the response to new therapeutic treatments. Comprehensive analysis of a tumor’s genetic profile is necessary to study the development of a cancerous growth, its progression and the emergence of drug resistance. NGS is used to help produce personalized pharmaceuticals, and thus offer the most effective prognosis.

Principles and process of NGS

NGS workflows involve four major steps—library preparation, cluster generation, sequencing and data analysis—with the first step, library preparation, being considered fundamental to the process.

During preparation, a library of nucleic acids is constructed by random fragmentation of the DNA or complementary DNA (cDNA) sample, followed by ligating adapted sequences onto the ends of the DNA fragments. This process is then followed by amplification and sequencing with polymerase chain reaction (PCR).¹ Target library quantification has been identified as a critical step, often determining the success of the entire run. Loading incorrect amounts of library DNA onto the NGS platform may result in low-quality data: too little will result in low cluster density and reduced sequencing yield, whereas too much may increase cluster density and result in poor-quality data. Either way, inappropriate amounts increase the probability of complete failure, which is timely and costly. For library quantitation, qPCR has become the technique of choice for delivering precise analysis of strand amplification in real time, allowing high repeatability and accuracy for pooling and/or loading into the flow cells.

Choosing efficient qPCR equipment to enhance NGS data

During qPCR, dsDNA is amplified using primers with complementary sequences to the previously annealed NGS adapter, a fluorescence signal is generated and the target strand is intercalated with DNA binding chemistry in the reaction mix. Capturing fluorescence in real time overcomes many inaccuracies associated with traditional endpoint PCR, including poor precision, low sensitivity and resolution and short dynamic range (<2 logs). qPCR does not require a gel, unlike traditional PCR, which eliminates risk of contamination.

Although qPCR is superior to traditional PCR, not all systems provide the repeatability and accuracy required for accurate NGS system loading. For primers to bind accurately, qPCR systems must allow uniformity across the entire heat block to ensure that all samples proceed through the reaction at the same rate. Precise temperature control is critical. The thermal accuracy of standard qPCR systems is around ±0.5 °C at the 50–60 °C range—enough variation to warrant concern about an insufficiency to precisely define the cluster density and risk of NGS failure if true library construction is under- or over-represented.

The importance of temperature precision is further highlighted by stringent Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.² qPCR systems have traditionally employed Peltier-heated thermal blocks to drive thermal cycling. Peltier-heated thermal blocks heat the entire block to a designated temperature, exceeding that of the reaction, before equilibrating to a desired plateau. The process is extremely energy-intensive, and run times can be long and inefficient due to the time it takes for wells within the block to reach the set temperature. This heating method also contributes to high thermal nonuniformity values and poor thermal ramp rates, meaning these systems are not suitable for high-performance applications.

Unlike its competitors, the recent advances in qPCR heating technology from PCRmax, which make use of a precise, electroformed 48-well hollow silver block that contains conductive fluid, ensure the highest uniformity of any block-based system. Via a single Peltier device, the block is heated and cooled with an agitator assembly consisting of two paddles driven by electromagnetic motors. During PCR cycling, the paddles move rapidly, circulating the fluid across multiple wells, allowing the block to achieve high ramp rates and reducing experiment time. Thermal stability of ±0.1 °C virtually eliminates thermal nonuniformity and prevents edge effects. The result is improved qPCR performance; tighter C_q; greater PCR efficiency; higher R2; and the ability to perform demanding, high-resolution melting. High-performance optical systems also allow the delivery of precise and sensitive fluorescence detection to facilitate all four color multiplex applications, while adaptive light-emitting diodes (LEDs) contribute to generating accurate data. Combined with innovative technology within the thermal cycler itself, the accompanying software is MIQE compliant and can be installed to as many PCs as required, allowing collection and analysis of the data. Overall, the Eco 48 provides a complete modular PCR system, capable of thermal cycling to data analysis. In combination with the thermal block design, the software comprises two elements—the Eco Control, which drives the unit’s setup and experimental runs, and the Eco Study, which provides data analysis. Although other commercially available PCR systems can complete thermal cycling, data collection and data analysis within a modular system, the Eco 48 block’s design ensures it is the most thermally accurate and uniform blockbased system. Use of the Eco 48 thermal cycler and accompanying software allows NGS library quantification to be performed while satisfying MIQE guidelines, without compromising speed, efficiency or cost.

Experimental

To define uniformity across the Eco 48 block, 48 replicate samples were subjected to a thermally demanding, high-resolution melt (HRM). In each of the 48 wells 10 × 10⁸ copies of template were distributed at a final volume of 10-μL. An Eco Seal plate (Bibby Scientific Ltd.) that was centrifuged for 1 minute at 1200 rpm was used for sealing. The entire 40-cycle protocol and associated HRM was completed in 43 minutes. Eco Study software was used to analyze the results to establish the C_q and T_m values for each of the 48 samples, as well as the degree of uniformity.

Figure 1 shows the baseline-corrected amplification plot for all 48 wells, with the graph clearly demonstrating precision of amplification across the entire plate. When the data was analyzed, an average C_q of 13.31 with a standard deviation of ±0.061 was calculated, and a %CV of 0.46% across the plate.

 Figure 1 – Baseline-corrected amplification plot showing the data from all 48 wells of the plate.

The T_m of the PCR product, achieved by running a melting stage following the amplification steps, is one of the best measures of block uniformity. The determined temperatures depend purely on the chemical composition of the product and are not reliant on the accuracy of external temperature probes. Amplified product is melted within the 75 °C to 95 °C range. Fluorescence is measured with every 0.1 °C of temperature change, which is sufficient to detect class IV SNPs with greater than 99% accuracy. Figure 2 shows the normalized melt curve.

 Figure 2 – Normalized melt plot showing the data from all 48 wells of the plate.

The T_m average across all 48 wells was recorded as 84.45 °C, with a standard deviation of ±0.058, and a %CV of 0.07% across the plate. Figure 3 shows a summary of the results.

 Figure 3a) and b) – Summary of C_q and T_m data from each of the 48 wells of the plate. The largest recorded variation across all 48 samples was 0.24 cycles, with a maximum range of T_m of 0.2 °C (84.3 °C to 84.5 °C) across the entire plate.

Conclusion

The Eco 48 is able to deliver complete heating uniformity and rapid cycles in 43 minutes, with the sensitivity to ensure ±0.1 °C uniformity across the whole block instantly after every temperature change. This makes it possible for library quantification to be successfully achieved within MIQE guidelines. The advances in technology, as displayed by the Eco 48, will allow consistent NGS runs to be completed, and further advances in genomics research to be made.

References

1. Grada, A. and Weinbrecht, K. Next-generation sequencing: methodology and application. J. Investigative Dermatol. 2013, 133, 1–4.
2. Bustin, S.A.; Benes, V. et.al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009, 55(4), 611–22.

Dr. Andrew Birnie, Ph.D., is business development specialist at PCRmax (a Bibby Scientific Company), Bibby Scientific Ltd., Beacon Rd., Stone, Staffordshire ST15 0SA, U.K.; tel.: +44 (0) 1785 812 121; fax: +44 (0) 1785 810 405; e-mail: [email protected]; www.bibby-scientific.com