In all sorts of science, the quality of a result depends on the starting material. That is particularly true with next-generation sequencing (NGS). When asked about the key challenges in sample prep for NGS, Thomas Wood, professor and NGS expert at the University of Texas Medical Branch at Galveston, says, “Quality and quantity—quality being the more important.”
For quantity, a researcher needs a way to measure the DNA in a sample before sequencing. For that, Wood recommends the Qubit technology from Thermo Fisher Scientific. A variety of tools, such as absorbance methods, also help scientists assess a sample’s quality.
New combinations of NGS and sample preparation improve a range of basic and applied research methods.
New combinations of NGS and sample preparation improve a range of basic and applied research methods. Moreover, improving and adapting the techniques make it possible to explore new questions with these methods.
Usual prep steps
“Despite the numerous types of NGS library preparation kits available in today’s market, most protocols follow the same key steps,” says Whitney Pike, application scientist at Advanced Analytical Technologies. That begins with fragmenting the target DNA into smaller pieces. “DNA can be fragmented either enzymatically or mechanically, such as by sonication or shearing,” Pike explains.
Next, the ends of the fragments get repaired and adapters are added. “These adapters are made up of unique barcodes, which allow for the multiplexing of several samples within one sequencing run,” Pike says.
Last, the polymerase chain reaction (PCR) amplifies the modified fragments. The resulting material is ready for NGS.
To improve sample preparation for NGS, Wood says that the most interesting recent advance is “using magnetic beads in the template assembly process.” In particular, he points out technology from New England Biolabs. He adds that this technology “will lead to greater utilization of robotics.”
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In all sample preparation for NGS, scientists need to ensure that the material is ready for NGS. “Researchers should perform quality control of their libraries following preparation using analysis instrumentation, such as our Fragment Analyzer,” Pike notes. “This will ensure that the library is of an acceptable size and concentration.”
Overall, the care in every step impacts the outcome. “From the start, it is important to use an input sample of good quality and of the correct size for the application chosen,” Pike explains. “Different extraction methods, tissue types, degrees of sample handling, and especially whether it was stored in formalin-fixed tissue can affect genomic DNA quality.” She adds, “To overcome these issues, the input materials, such as genomic DNA, should be checked for quality or integrity using an electrophoresis method.”
SMRT steps
At the end of 2017, Pacific Biosciences released its SMRTbell Express Template Prep kit. This is designed to prepare samples for PacBio’s single molecule, real-time sequencing (SMRT).
For SMRT sequencing, Paul Kotturi, director of product management at Pacific Biosciences, explains: “As we typically sequence native DNA, samples are taken through a simple set of steps to remove DNA damage, repair the ends of the insert and ligate on hairpin-loop adapters, called SMRTbell adapters.” Those adapters turn fragments into circular molecules.
“Once the SMRTbell templates are generated, a sequencing primer is annealed at one of the SMRTbell adapters and a SMRT Sequencing DNA polymerase is bound,” Kotturi explains. That complex is ready for sequencing, and PacBio reads are usually long, more than 30,000 bases.
Image: Special cells contain samples for single molecule, real-time sequencing. Image courtesy of Pacific Biosciences.
With the SMRTbell Express Template Prep kit, the workflow, says Kotturi, “requires a total of about three hours with much less hands-on time.” This protocol uses just one tube, and researchers only add to it. In pointing out the key advantages of this workflow, Kotturi mentions: minimizing sample mishandling, because samples stay in a single tube; minimizing potential DNA damage, because of fewer sample manipulations; and the reduced opportunities for sample loss produce a higher yield of DNA.
Other applications
As forms of sequencing advance and improve, so do uses of NGS. This also depends on evolving sample-preparation techniques. “One of the most interesting recent advancements in sample prep for NGS is single-cell library preparation,” Pike points out. “Being able to make a library from individual cells enables scientists to measure gene-expression differences over time from one cell to the next.” She adds, “This capability is critical for being able to understand tumor heterogeneity and how cells grow and mature.”
At Ohio University’s genomics facility, director William Broach uses NGS in various ways. “Our most common use for NGS is for RNAseq to explore gene-expression changes in both prokaryotic and eukaryotic systems,” he says. “We also perform a large number of amplicon sequencing particularly focused on exploring the microbiome of different organisms.” In this work, says Broach, one of the key advances in sample preparation is the ability to use extremely small amounts of RNA or DNA. “In the realm of single-cell prep,” he says, “this really allows researchers to understand what is happening at the cellular level rather than being required to pool cells and thus getting an average of gene expression or average genomic DNA profile.”
For non-invasive prenatal testing (NIPT), scientists can use cell-free DNA (cfDNA) libraries. “Sequencing-based NIPT continues to grow in prevalence as it becomes clear that cfDNA isolated from the mother can provide important information on fetal health,” Pike says. “The number of cfDNA samples used for liquid biopsies is expected to rise dramatically in the near future.” For now, scientists need to work more on these processes. “Understanding how the cfDNA quality affects library preparation is still in its infancy,” Pike adds.
For any application of NGS, the outcome depends on the sample-preparation steps and performing them as accurately and consistently as possible. Plus, the results must be confirmed. The approach to preparing a DNA sample will depend in part on the method of NGS that will be used. Likewise, the specific question at hand can impact the preparation technique.
Ultimately, the outcome of sample preparation determines the potential for usable sequencing. Without preparing the proper sample material, the investment in NGS goes to waste.