Expert Tips to Improve NGS Sample Prep

Despite the huge advances in next-generation sequencing (NGS) for generating genomics data, getting to the results can be time-consuming and labor-intensive. Sample preparation can create a time and labor bottleneck, which gets even more complicated when working with damaged or degraded samples. A few suggestions from experts, though, can really speed up and simplify the sample-prep process.

Two of Hamilton Company’s experts—Sha Liao, senior market segment leader for genetic screening, and Ryan Ghan, market segment leader for biotechnology—teamed up to provide information, and they wrote: “If we think of DNA as a string of beads, then each bead represents a nucleotide. The goal of NGS is to figure out which bead goes where along the string.”

Let’s find out the best ways to prepare samples to get that information.

Obstacles that will be encountered

A variation in a sequence could be a natural variant or the result of the NGS workflow. Various steps in sample preparation could be at fault if a variation is due to sample processing. “As assays become more miniaturized to suit high-density formats, imprecise pipetting may arise to impact data as well,” the Hamilton experts noted. “If labs find that, after a lengthy NGS process, they have bad data, they have to start again, which consumes valuable sample and also increases reagent and labor costs per sample when the process is rerun.” So, sample preparation for NGS should be consistent and repeatable.

“As we remove these external sources of inconsistency and variability throughout the workflow, we can be more assured of true results,” the Hamilton duo pointed out. “As the sequence is read over and over again, repeatability in the depth of a given nucleotide or sequence is what provides confidence that a given variant—or bead on the string—is truly representative of the sequence.”

When asked about the biggest challenges in NGS sample preparation, Michael Beer, product manager for liquid handling and automation at Analytik Jena, provided a useful list, including:

• throughput
• many sample-prep steps
• hands-on time
• human error
• cost per sample
• cooling of samples and reagents
• finding the right kit for a specific application

To delve into the details, Giron Koetsier, development scientist II at New England Biolabs, provided some specific challenges for sample preparation upstream of long-read sequencing. “Extracting high molecular weight DNA quickly and easily has historically been a bottleneck,” he says. “Phenol extraction is probably the most tried-and-true method, but has several drawbacks, which include being a lengthy process that involves working with hazardous materials.” He summarizes that method as: “quite inconvenient, unpleasant, and laborious.”

Even handling high molecular weight DNA gets complicated. “It is viscous and does not go into solution easily or homogeneously,” Koetsier said. “Because of this, getting accurate quantitation and purity assessment of very high molecular weight DNA is still very challenging and leads to variation in sample input amounts.”

Despite the challenges in extracting high molecular weight DNA, some applications benefit from using longer DNA fragments. For such applications, the DNA extraction process must isolate large molecules and not short ones. “The presence of shorter fragments can be a result of the extraction process—such as lysis conditions, use of bead beating, or magnetic beads—or a result of low-quality starting materials, such as degraded ones,” Koetsier said. Consequently, it is important not only to start with high-quality input material, but to use an extraction workflow that minimizes nuclease activity and DNA shearing.

Finding the right fix

These experts revealed some of the obstacles that scientists face in preparing samples for NGS, and they also suggested some solutions.

“Automation is a must-have when it comes to standardizing complex NGS sample-preparation workflows,” noted Liao and Ghan. “Automated liquid-handling workstations offer increased reproducibility and precision compared to manual pipetting.” They added that “many automated workstations support multiple library protocols to increase flexibility in the laboratory.”

Beer agreed with the benefits of adding automation, and he described various ways to do it, “ranging from the semi-automation of processes like nucleic-acid extraction, clean-up steps, size selection, adapter ligation, enrichment with an automated liquid-handling solution, up to fully automated solutions for the execution of the whole library prep workflow.” He added that automation could use a workflow from a cloud-based solution that “empowers the user with advanced data analysis and sharing of data worldwide.”

For longer-read applications, Koetsier recommended replacing phenol extraction with newer, innovative solutions, such as the new approach from New England Biolabs that “uses large glass beads coupled with an optimized lysis chemistry to produce highly pure, intact, and very large DNA fragments in a very short amount of time.” He added, “Using this approach, DNA into the mega-base range can be isolated from cells and other samples that have been historically challenging, like tissues and blood.”

Future fixes

Even with the advances noted by these experts, more improvements are needed in sample-prep technologies. For example, “certain bacteria and plants are difficult to lyse, which makes isolating good quality high molecular weight DNA challenging,” Koetsier said. “Advances in extraction technology for these challenging sample types is still widely needed and sought after.”

In general, a range of improvements lies ahead for NGS sample preparation. As Beer promised: “Standardized solutions within automation will become more and more important.”