High-quality sequencing for genomes - harnessing the power of long-read sequencing
NGS is in widespread use throughout the life sciences sector, but the commonly used short-read sequencing methods are inherently ambiguous and prone to GC base pair bias. Pacific Biosciences (PacBio) offers longer reads, uniform coverage, and high accuracy using its Single Molecule, Real-Time (SMRT®) technology and Sequel® Systems. This proprietary approach is ideal for advanced genomics, full-length transcript sequencing and epigenetics, and has proven invaluable for labs researching the SARS-CoV-2 virus, helping them to understand the genetic characteristics of the virus itself and to characterize human immune responses to COVID-19.
Long-read sequencing for improved analysis
PacBio’s Sequel instruments sequence DNA directly in SMRT Cells packed with zero-mode waveguides (ZMWs) – essentially very small reaction vessels. A polymerase with the pre-bound DNA template is anchored at the bottom of each ZMW, and fluorescently-tagged nucleotides are introduced to begin sequencing. The ZMW is illuminated from below, and the attenuated light enters the lower 20-30 nm of each ZMW, exciting the fluorophore as the polymerase incorporates each base into the native strand. This allows the system to effectively ‘eavesdrop’ on the process of DNA synthesis in real time. Marty Badgett, Senior Director of Product Management at PacBio, explained the advantages of this approach: “The main benefits are the long reads, uniform coverage and high consensus accuracy. This accuracy is due to low systemic error and the random nature of read errors. For instance, with approximately 10-fold coverage we can achieve 99.9 % accurate reads, as random errors wash out of the data.”
The company’s original system – the PacBio RS II – had 150,000 ZMWs per SMRT Cell, equating to about 55,000 reads, and could process up to 16 SMRT Cells per run. However, with NGS becoming ever-more prevalent and cost effective, throughput was still a limitation for many customers, leading to the development of the Sequel System in October 2015. With this platform, each SMRT Cell contains one million ZMWs, offering a seven-fold improvement in throughput. Paz Yogev, Senior Staff Mechanical Design Engineer, commented: “When we were developing the Sequel System, we wanted to incorporate a number of additional liquid handling features that the pipettor used in the PacBio RS II simply couldn’t offer. We researched a number of solutions, and chose the Cavro® Air Displacement Pipettor (ADP) from Tecan for its combination of performance, accuracy, cost and advanced features, including automatic tip detection and ejection, and liquid level detection.”
Marty added: “As soon as we launched this platform, we began working on our next generation chemistry and a higher density SMRT Cell containing eight million ZMWs for what would become the Sequel II System. The Sequel was already developing a strong reputation for reliability at this time – in part thanks to the pipetting performance of the Cavro ADP – so we decided to make as few changes to the hardware as possible. This would not only ensure the platform’s continued reliability, but would also allow us to launch the system into the market faster.”
Fine tuning performance
“Our aim was to refine the instrument’s protocols and further improve the overall robustness of the system,” Marty continued. “For example, we introduced some self-testing and analysis functions for the ADP, allowing the system to identify any deterioration in pipetting performance and, crucially, enabling remote diagnostics if a customer encounters a problem. We haven’t had any concerns about the ADP’s performance, and this remote monitoring can help to reduce the number of field engineer visits to customer sites to resolve issues that may have nothing to do with the instrument itself. This was very easy to implement using the ADP’s built-in diagnostic functions, without us needing to alter the instrument hardware.”
“We have been working with the Cavro ADP for a number of years now, and it has been really good to us,” Paz commented. “From the production of the first Sequel System to the latest improvements to the Sequel II System, it’s been very reliable. Our field service engineers respond to any problems encountered by customers, and we very rarely need to reach out to the Tecan team for extra support, but we know it’s there if we need it. This quick response is equally important for our R&D. As a relatively small company, we try to move extremely fast to meet market requirements, and our relationship with Tecan is crucial to achieving very short instrument development cycles. We always receive a very high level of technical support if we have any questions.”
Broad applications for COVID-19
NGS has been at the forefront of research into SARS-CoV-2, providing valuable information to labs developing molecular or immunoassay tests and vaccines, as well as offering insights into the diverse immune responses seen in vivo. Dr Meredith Ashby, Director of Market Strategy for Microbial Genomics, Immunology and Infectious Diseases, explained: “When it became apparent that the COVID-19 crisis was going to become a global pandemic, over 50 of our customer sites – many of them not virology labs – expressed an interest in working in this area. To give our customers the best possible tools to tackle this global emergency, we began testing and validating the various sequencing protocols that were emerging.”
“As the pandemic has progressed, it's become clear that the novel coronavirus has a relatively low mutation rate for an RNA virus, but the range of immune responses – and therefore the severity of disease – among patients is enormous. As a result, many of our customers have turned to sequencing to investigate this variation. One of the strengths of the Sequel II system is that it can perform highly accurate long reads (HiFi reads), offering sequencing reads of up to 20 kb in length. This has been a huge advantage for looking at the highly polymorphic regions of the human genome involved in the immune response, as well as the very diverse range of antibodies produced in response to the virus, which we hope will contribute to the rapid development of an effective vaccine. The whole scientific community is obviously committed to this, and it’s nice to be able to do our part,” Meredith concluded.