NGS Sample Preparation Taking the Pain Out of Painstaking

Although next-generation sequencing (NGS) keeps giving—exploring the human genome and revolutionizing our knowledge of health and disease—it needs to take a little, too. At the very least, NGS requires that attention be devoted to sample preparation. Currently, workflows can be complicated, costly, and challenging for the analysis pipeline.

We spoke to several leaders in the field about how the NGS industry is responding to the need for NGS users to enhance productivity while maintaining economy. One approach is focusing on streamlining workflows and reducing the number of consumables required. Another is utilizing novel methods to assess the methylation status of the genome in a single analysis. Because magnetic beads offer an avenue for sample preparation, special engineering is enhancing yield and assuring the tight size distributions needed for NGS. Further, CRISPR-based depletion solutions are aiming to maximize read depth for targeted areas. Finally, the bottleneck of manual sample preparation may be giving way to a push-button, fully automated future.

Next-generation workflows

“As with all exciting technologies, surprises and challenges are inevitable,” observes Betsy Young, PhD, senior product marketing manager, New England Biolabs (NEB). Young includes NGS sample preparation among those exciting but vexing areas.

“When someone says ‘sample prep’ or ‘library prep,’ they’re describing a process by which their sample of nucleic acid is made compatible with any of a variety of sequencing platforms,” she continues. “If library prep is subpar, you may see low library yields, uneven coverage, unexpected biases, low library complexity, high levels of duplicates, and even chimeras. Unfortunately, most of these issues become apparent only after a costly sequencing run.”

This results in a loss of time and money. To enhance more efficient workflows, NEB has several new offerings. Young remarks, “2024 marks NEB’s 50th anniversary as a company that supports scientists engaged in all areas of molecular biology. For the last 15 years, NEB has been instrumental in enabling scientific discovery involving NGS with its suite of NEBNext DNA and RNA library prep kits, modules, and reagents.”

In continuing that tradition, the company released its next-generation workflows for sample prep. “Our newest and most exciting update to the NEBNext portfolio is the recent release of NEBNext UltraExpress library prep kits for DNA (fragmented or intact) or RNA,” Young says. “These kits represent the next generation of NEBNext’s faster, more flexible workflow, with exceptional time savings and performance metrics.”

According to Young, these kits streamline workflows and require significantly fewer consumables: “Our NEBNext UltraExpress FS DNA Library Prep Kit workflow uses 32% fewer tips and 50% fewer tubes than our previous solution, while the NEBNext UltraExpress RNA Library Prep Kit workflow takes half of the time. In fact, users can go from total RNA to library in 3.5 hours when the RNA library prep kit is combined with the NEBNext Poly(A) mRNA Magnetic Isolation Module and its Express protocol option.”

Looking ahead, Young forecasts, “As NGS continues to be more widely adopted and is increasingly relied upon as an essential method in research, diagnostic, and clinical applications, the importance of robust and reliable library prep will only increase, as will the need for more streamlined workflows that make NGS more accessible and higher throughput.”

Enhancing efficiency

Illumina points out that many NGS sample prep workflows can be complicated, inefficient, and require scientists to stitch together different data types using custom tools to gain insights. “This requires that a substantial amount of time and resources be spent on executing the experiment and the analysis pipelines rather than finding the answers,” relates Cande Rogert, PhD, vice president, global head of advanced science. “On top of this, NGS sample prep can be a cumbersome and time-consuming process that limits broader adoption and extends turnaround times.”

The company recently announced two sample prep innovations for increasing customer insights while simplifying NGS workflows. “The first innovation is the five-base genome,” Rogert says. “This technology will give customers both variant and epigenetic information from a single library prep workflow.”

The fifth base is a methylated form of cytosine (5-methylcytosine). A simple additional step in library prep allows users to directly detect methylated cytosines. Scientists will be able to gain insights into methylation status of a genome without having to perform separate sample prep workflows, sequencing runs, or data analyses.

“The methylation accuracy of our approach matches today’s industry-leading standards, and variant accuracy surpasses existing solutions,” Rogert continues, “And all of this can be achieved at a low cost in a single library prep and sequencing workflow without the need to stitch data together.”

The company’s second innovation is its comprehensive genome. “This product has two main advantages,” Rogert asserts. “First, it eliminates the manual library prep steps ahead of the sequencer by integrating them onto the surface of the sequencing flowcell. Second, our comprehensive whole genome gives customers a more complete genomic readout. This innovative method enables coverage of challenging genomic regions and complex structures, such as large structural variants, repeat regions, and phasing information, while maintaining small variant calling accuracy.”

Rogert adds that the field can look forward to additional improvements: “We are just at the beginning with many exciting discoveries to come within the fields of genomics, multiomics, and beyond. We believe NGS sample prep workflows and instrumentation will continue to play a central role in these discoveries. We have a responsibility to continue to deliver new innovations and advancements to streamline current processes for researchers and advance insights. We feel the best of genomics is yet to come!”

Reducing complexity

During NGS sample prep, the use of multiple reagents and manual interventions can reduce economy and productivity. “Further, single-cell analyses can be hampered by low throughput that challenges its use for clinical applications such as cancer diagnostics and personalized medicine,” says Andrew Gane, diagnostics and genomics technology leader, Cytiva.

According to Gane, Cytiva’s main focus in the area of NGS and NGS sample prep is to enhance the efficiency, accuracy, and scalability of workflows. “We aim to reduce costs associated with NGS, making this powerful technology more accessible across research, clinical, and industrial settings,” he explains. “Our focus is also on supporting precision medicine, particularly in areas like oncology, reproductive health, and infectious diseases.”

Gane adds that the company provides a number of cost-saving measures for NGS sample prep: “We offer technologies and solutions to support sample and library prep for today’s high-throughput sequencing demands including single-cell analysis. Our magnetic bead–based kits simplify sample prep, reduce hands-on time, and improve the reproducibility of results.”

Specifically, the core shell design of the company’s Sera-Mag Speedbead magnetic beads was engineered for the high yields and tight size distributions needed to enhance NGS efficiency and consistency. As an added benefit, the amount of required starting material for bead use is reduced, resulting in greater efficiency and lower costs.

“The future of NGS will likely see the integration of AI-driven analysis and further miniaturization of sequencing platforms,” Gane declares. “These advancements will continue to drive down costs and expand the reach of NGS into new fields, making personalized medicine more accessible on a global scale.”

CRISPR-based depletion

One of the critical needs for efficient NGS is how to optimize sample prep so that read depth for targeted areas is maximized while minimizing costs. This process can be assisted by using depletion studies to rid the sample of contaminants.

“In gene expression studies, a significant proportion of the libraries that are sequenced (up to 50%) are noninformative,” says Pedro Echave, senior manager, global business segment, Revvity. “They’re just filtered out bioinformatically during secondary analysis. By removing noninformative molecules before sequencing, we enhance the complexity of the library, increase the detection of lower frequency variants, or remove the existence of dropouts for specific regions or genes of interest.”

Echave also notes this process can greatly lower costs because “researchers do not have to sequence through all the noninformative RNA in their samples to get the information that they would like to obtain.”

In order to remove noninformative RNA, Revvity scientists are turning to CRISPR-based technologies. “A recent innovation is the incorporation of CRISPR-based depletion solutions in our portfolio for a variety of RNA-seq applications, including single-cell analysis, human transcriptomics, and metatranscriptomics,” Echave relates. “The main advantage of CRISPR over other technologies available in the market is that it is extremely intolerant of mismatches—it’s highly specific even in comparison with targeted approaches. It’s also very easily incorporated into most existing workflows, and it is compatible with short- and long-read sequencing.”

Echave says that Revvity is focused on “streamlining and automating” the entire NGS sample prep process: “Our company offers end-to-end workflows that include sample homogenization, nucleic acid extraction, library prep, and quality control. Revvity also provides specialized solutions for various applications, such as newborn screening, small RNA sequencing, and RNA depletion.”

Push-button sample prep

What if sample prep for NGS were as easy as pressing a button and walking away? Volta Labs says that scientists can do just that with the company’s new Callisto Sample Prep System. “While sequencing technologies have made huge advancements, sample prep is largely done manually,” says Udayan Umapathi, founder and CEO, Volta Labs. He stresses that new solutions are needed to solve the sample prep bottleneck.

Resolving this problem with automation wasn’t easy, though. Umapathi reports, “This required developing a technology platform that is flexible enough to work with various sample types and chemistries while maintaining quality that would span many technical disciplines—fluidic dynamics, thermal engineering, molecular biology, etc.”

Enter the Callisto Sample Prep System, a sequencer-agnostic benchtop system. “It enables a push-button, walkaway sample prep experience,” Umapathi asserts. “This enables all labs—labs already using sequencing automation, and those new to sequencing—to free up resources and not rely on skilled labor to maximize throughput.”

How does it work? The system manipulates electric and magnetic fields to place little droplets on a surface. These droplets contain DNA, RNA, enzymes, and other biochemistries, or they can be raw samples such as blood or saliva. The droplets are then manipulated and split in various ways followed by heating to specific temperatures.

Umapathi says that the Callisto Sample Prep System is the first of its kind in providing an “app-based solution” to customers: “Preloaded apps are ready to run from day one—no method optimization required. There is no need for a dedicated user or operator, eliminating method development costs. Also, apps are compatible with most commercial sequencers for DNA and RNA samples.”

“Innovations such as the Callisto Sample Prep System are likely to expand the use of NGS,” Umapathi predicts. “We expect broader NGS adoption, especially in emerging countries. From a Volta standpoint, sample prep will become increasingly standardized and streamlined. Just like sequencing, it will become a push-button task. This will mean overcoming the big hurdle that exists because of the skilled labor shortage and propelling greater adoption of sequencing technology.”

The post NGS Sample Preparation Taking the Pain Out of Painstaking appeared first on GEN – Genetic Engineering and Biotechnology News.

Source