Improving NGS Library Prep with Automation

One of the best ways to ensure success in next-generation sequencing (NGS) is to begin with a high-quality NGS library. Manual preparation of NGS libraries involves many repetitive high-precision pipetting steps of small volumes. The mind-numbing repetition of such work can raise the error rate when performed by human hands, but is perfectly suited for automation. Here we detail some of the many benefits of automating your NGS library prep workflow, and some expert advice for switching over.

The benefits of automation

When performing NGS sample prep manually, the higher likelihood of errors or inconsistencies can be problematic. One example is the possibility of sample-to-sample contamination. “Aside from eliminating contamination issues, automated processing also alleviates the need for lengthy and labor-intensive sample handling by a human,” says Ryan Ghan, Market Segment Leader in Biotechnology at Hamilton. “This is especially true for genomic core sequencing facilities and diagnostic laboratories, where high throughput and/or rapid turnaround time are of the utmost importance.”

A properly automated workflow gives more consistent results than the work of human hands and brains. Because of this, workflows that are the best candidates for automation are the especially tedious and/or complicated ones—these are most vulnerable to human error in the face of flagging concentration, boredom, distraction, and other causes of inconsistencies. “With automation, the primary benefit is the recouped cost of lost opportunity,” says Rupert Yip, VP of Marketing and Product Management at Tecan. “The researcher is freed up to do more scientifically creative work, rather than the mundane work that a machine can perform.” Automation may also be more scalable and cost-effective “because machines don't call in sick or won't complain about working 24/7,” adds Yip.

With lower error rates, automated workflows reduce human risks of chemical exposures and repetitive stress injuries, and increase throughput. “With large labs, the benefit of increasing throughput is often top-of-mind as manual approaches can typically be the rate-limiting bottleneck within the workflow,” says Henry Shu, Senior Product Manager in the Genomics Group at Agilent Technologies.

Automation may offer more versatility as well. Open platforms have the ability to automate NGS library prep workflow from practically any manufacturer. “Hamilton offers open platforms so that users may continue to use their preferred assay chemistry, implement new protocols or modify existing ones, or even run multiple chemistries on one convenient liquid-handling workstation,” says Ghan. “This versatility can be a critical factor as laboratory needs evolve over time.”

Deciding to automate

Whether to choose automation will depend on multiple factors, including overall cost, quality, turnaround time, and throughput. It’s a good bet that automation will reduce cost and increase quality (though you should run trials to convince yourself first). “Automation results should be equivalent [in quality and accuracy] to results from the manual process—ensure there is data to prove this,” says Fink. “If a researcher is hesitant to walk away for hours and let the automation run unattended, there are cloud services available, such as Revvity's PKeye™ workflow monitor, which monitors instruments in real-time and will send instant email or text messages and video clips of status and alerts.”

But what about time and throughput? “With few exceptions, a human will likely complete a complex workflow faster than a machine, because machines need time to move the robotic arm around, pipette slowly (for accuracy), and move the arm to dispense used tips,” says Yip. The quicker movements of human arms and fingers make better time through a multi-step workflow than do robotic arms.

But automation is more likely to win in throughput. “As automation can work 24/7/365, it can process more samples over a week than a pair of human hands—similar to using a fighter jet versus a passenger jet to ferry 300 people from New York to London,” notes Yip. “One may be faster but the throughput of the other is indisputable.”

Consider also the difficulty level of your intended library prep. Preparing mRNA libraries, for example, can be particularly difficult due to the ease of RNA degradation. “A scientist who rarely performs mRNA library preps can expect failure rates as high as 50% when performed manually,” says Yip. “If an equivalent automation solution can consistently produce successful libraries at say >90% of the time or across >90% of input samples, then it behooves the researcher to pass all mRNA samples through automation rather than risk failure using manual methods.”

Automating quality control

Quality controls, an important factor in library prep, should be included in the automated workflow. For example, Tecan’s platforms check the sample purity, and DNA library size and density. “Tecan Genomics’ proprietary library preparation chemistry also incorporates novel chemistries that allow simultaneous QC measuring and quantification into one step, thus further reducing workflow time,” says Yip.

Hamilton’s systems can automate the preparation of plates for quantification by qPCR, or integrate with plate readers for absorbance or fluorescence measurements. “Other measures that we offer to increase quality control while reducing human contact include barcode scanning to facilitate sample and reagent tracking and real-time monitoring,” says Ghan. “By creating a chain of custody for each sample, users have traceability of volume transfers through each sample processing step.”

Ghan also notes other factors in play for proper QC, such as defining liquid class parameters for each reagent. This allows the pipette channel plungers to optimize the precision of their movements during liquid transfers. “Hamilton offers pre-programmed methods that are specially optimized in collaboration with leading NGS chemistry providers,” says Ghan. “This reduces time and effort that users would otherwise spend on individualized method development.”

Agilent offers several automated electrophoresis instruments for library QC, including the TapeStation, Fragment Analyzer, Femto Pulse, and Bioanalyzer systems. “The sample quality metric determines the recommended fragmentation time and PCR cycle numbers for optimal results,” says Shu. “Agilent offers automated protocols with the Magnis NGS Library Prep System, the Bravo NGS, or Bravo NGS Workstation which are designed with convenient sample QC checks on one of our automated electrophoresis instruments.”

Automating the QC protocols is important for the same reasons as for automating library prep protocols—but even more so, according to Arvind Kothandaraman, Managing Director of Specialty Diagnostics at Revvity, because “after the library preparation, the samples have become more precious.” He suggests incorporating normalization beads during library preparation to reduce QC steps. “Revvity's NEXTFLEX^® normalization beads provide a consistent mass and sequencing cluster density for all samples in a library pool, regardless of input amount,” says Kothandaraman. “This shaves off up to three hours in the high-throughput quantification and pooling preparation.”

Revvity's LabChip^® GX Touch™ Nucleic Acid Analyzer can also automate library QC tests. “Based on modern microfluidic technologies, it can generate high-quality digitized DNA and RNA quantitation and sizing data within 30 seconds with minimal input requirements,” says Kothandaraman. “With the unique sipper microfluidic chip design, the system can measure 384 samples in one experiment preparation.”

Tips for switching over

Once you’ve decided to automate your library prep workflow, these expert tips might ease the process. Ghan recommends physically mapping the components involved, including reagents, consumables, equipment, and people. “This map can help to uncover pain points and guide decision-making during the transition to an automated system,” he says.

Consider the batch size of your automated library prep workflow, in order to balance dead volume costs associated with reagent consumption, and turnaround time. “If there are not enough samples in the batch to spread out the cost associated with the dead volume, the cost per sample will go up compared to manual workflow,” says Shu. “On the other hand, if the user batches up samples to reduce the cost per sample, it will impact the turnaround time.”

Also consider the level of automation, whether you need partial or full. “Fully automated NGS library prep solutions, such as Revvity's Sciclone^® G3 NGSx iQ™ workstation, offer full turnkey, walk-away automation for NGS library prep including fully automated liquid transfers, thermal cycling and bead clean-ups,” says John Fink, General Manager of Automated Liquid Handling at Revvity, noting that using an automation provider with demonstrated library prep solutions is the best way to get a new automation system set up and running quickly.

To minimize materials, miniaturization of reaction volumes may be an advantageous route. This is possible on low volume liquid-handling platforms, such as the Revvity FlexDrop™ iQ non-contact nano dispenser, and Sciclone G3 NGSx HT workstation. “There is a growing trend to reduce the cost of next-generation sequencing,” says Fink. “Backed by standardized and tested miniaturized protocols, these instruments are capable of miniaturizing NGS reactions at up to 1/10th volume, saving labs considerable money.”

If you decide to take the automated plunge, consider it the beginning of a beneficial process. “Increasing lab efficiency is a long-term improvement that begins after switching from manual to automated NGS library prep,” says Shu. “Investing in automation is an investment in your team and pivots your lab into the future.”