Next-generation sequencing (NGS) library preparation, an important first step in the NGS workflow, used to be a difficult, labor-intensive process. Improvements are being made, but it remains important not to cut corner as heeding the adage “garbage in, garbage out” here will support high-quality NGS sequencing results. Today companies offer kits for NGS library prep that streamline the process, and many kits are automation-compatible. This article looks at some available kits as well as the benefits of switching to automated NGS library preparation.
Types of kits
The importance of high-quality NGS library preparation lies in the fact that this step changes sample DNA or RNA into a form that the NGS sequencing instrument can read. This is accomplished by fragmenting the nucleic acid into smaller pieces, and attaching adapter sequences onto the fragments’ ends. Sometimes unique barcodes are also added to allow for sample indexing, which is particularly important for high-throughput preparations.
GE Healthcare provides core materials, such as their Sera-Mag magnetic beads, for a lot of NGS library prep kits sold by third parties. In addition, GE offers some ready-to-use kits for scientists. “For example, we recently launched our Sera-Xtracta range of genomic DNA kit and cell-free DNA isolation kits,” says Andrew Gane, genomics and diagnostics strategy and technology manager at GE Healthcare. “The cell-free DNA isolation kit aims to deliver improved sensitivity from liquid biopsy samples, especially in sequencing and digital PCR analysis of cell-free DNA.”
GE also offers solutions for amplification such as GenomiPhi and TempliPhi to generate sufficient starting materials for downstream analysis as well as sample preparation tools such as Sera-Mag oligo(dT)-coated beads used in NGS library prep for pulling out mRNA prior to RNAseq. “It is recommended in several third party protocols, and we have a number of other OEM partners that actually use it in RNA sequencing library kits,” says Gane. For PCR cleanup within the NGS workflow, to purify DNA or RNA, GE’s carboxyl Sera-Mag beads are used by a number of companies that provide magnetic bead based PCR cleanup and size selection reagents for NGS library prep. GE also offers their own ready to use solution with their Sera-Mag™ Select PCR clean-up and size selection kit.
Kits incorporating features such as automation readiness, sample indexing, and targeting to specific sample types or nucleic acids, are available from multiple suppliers. For example, Revvity offers a complete workflow for NGS library preparation including liquid handlers, automation-ready kits for DNA, RNA, small RNA library preparation, and quality control of libraries. NEB’s NEBNext® line of automation-compatible NGS library prep kits also supports multiple sample types and nucleic acids.
Agilent offers kits that are compatible with their two platforms for automated NGS library prep. The SureSelect XT Low Input, XT, and XT2 Kits are compatible with the Bravo NGS Workstation, while the SureSelect XT HS Kit is compatible with the Magnis platform. “The Bravo NGS Workstation is built on the Bravo automated liquid-handling robot preconfigured for library prep and enrichment when using NGS protocols,” says Viresh Patel, senior director of marketing for genomics, in the diagnostics and genomics group. The SureSelect XT Low Input Kits for the Bravo are optimized for intact or degraded FFPE samples with as little as 10 ng DNA. The SureSelect XT Reagent Kits enable post-capture pooling of libraries, while the SureSelect XT2 Reagent Kits allow pre-capture pooling of libraries.
Agilent’s Magnis system is a benchtop instrument for fully automated NGS library preparation, and uses the SureSelect XT HS Kit. It can handle intact or degraded FFPE samples, incorporates automated barcode checking, and requires little experience to run due to self-checking routines and an on-board wizard.
An important step in the preparation of NGS libraries is selecting for the size of DNA or RNA fragments. Several companies offer tools for fragment size selection and reaction clean-up, such as MACHEREY-NAGEL’s NucleoMag® NGS Clean-up and Size Select beads. “Our beads can be combined with the diverse [NGS] library preparation kits available on the market, as well as with diverse automation platforms,” says Marko Gogala, product manager in bioanalysis at MACHEREY-NAGEL. “This flexibility and easy integration into existing manual and automated workflows is a major advantage of the NucleoMag NGS Clean-up and Size Select beads.”
Switching to automation
The benefits of switching from manual to automated NGS library prep are many. Automated prep is faster, more efficient, less prone to human errors (such as during pipetting), less susceptible to contamination, and more reliable and consistent. In addition, incorporating automated quality control (QC) tests can help to optimize the workflow (see next section). Keeping a few points in mind when switching can help to smooth the process.
One of the easiest methods for automation is with magnetic beads, notes Gane, because you just need to use a multi-well plate format and a magnetic plate, without the need for difficult-to-implement solutions. “If I was starting to develop an automated process, I’d go with an open system with a magnetic bead based approach,” he says. Gogala also recommends that researchers use an open rather than closed automation platform. Closed platforms—those tied to a single supplier—may cost less initially, but consumables can become more expensive with time. “In choosing an open platform, the user keeps their options open and can combine the best solutions from diverse suppliers,” he says. “Additionally, open platforms are easier to adapt in case of delivery bottlenecks or changes in protocol.” Gane agrees with this sentiment. “I think that if you buy an expensive piece of equipment for automation, you should really be able to do what you want with it,” says Gane. “You shouldn't be restricted to buying reagents from that same company.”
Patel recommends that researchers think about sample numbers when considering a switch to automation. For example, higher-throughput labs would be better suited to a system like the Bravo, which processes 96 samples at once. “For mid- to low-throughput labs, the Magnis could be the better choice, [as it] processes eight samples at a time, ” he says. Level of automation is also important to consider. “If you want a fully automated workflow, the Magnis system is ideal as it is a touch button walk-away system,” Patel notes.
Optimizing automated workflow
Once you have made the leap into automation, it’s important to consider how best to optimize your protocols. “Optimization is key—not everything can be done using lab automation in the same way things are done manually,” says Fiona Stewart, NGS product portfolio manager at NEB. “The nuances of pipetting operations, temperature control for enzyme incubations, and timing may require creative approaches and careful optimization to ensure that automated results match those obtained manually.”
Quality control of NGS libraries is another important tool for optimizing sequencing results. “For the best results, QC should be performed on several steps including incoming raw material, intermediate steps, and final library,” says Patel. Agilent offers tools for automated electrophoresis of genomic DNA that can QC raw material, such as their Fragment Analyzer, TapeStation, and Femto Pulse systems. These tools can empirically score FFPE or otherwise degraded DNA according to quality.
Checking the quality and quantity of NGS libraries before sequencing is highly recommended, notes Arvind Kothandaraman, director/general manager of next-generation sequencing, applied genomics, at Revvity. “Library quality is a critical factor in next-generation sequencing [as] a low-quality library can skew results, and affect reliability of reads and reproducibility,” he says. Visualizing the library using electropherogram traces can give a qualitative look at library size and relative abundance of adapter dimers. Libraries can be rapidly quantified by fluorometric analysis or qPCR. Even though qPCR assays are more expensive and laborious, “they ensure the most accurate library quantitation as only doubly ligated molecules are measured, [though] manual assay set up is prone to contamination and user errors,” he adds.
NEB generally recommends assessing the molecular size distribution and overall yield after the NGS library prep process. In the end, “sequencing results are in many cases required to confidently ensure performance is optimal, as sequencing can most accurately assess the complexity of library molecules available for analysis,” says Stewart. Taking prudent steps to optimize your NGS library preparation and maintaining quality protocols should ensure that you see the proof of your hard work in excellent sequencing results.