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Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
This article walks readers through the reasons why these steps affect sequencing data, which samples are the ones where researchers have to be the most careful, and the latest advances in reagents and protocols that help reduce variability from those three steps: collection, preservation, and sample preparation.
Figure 1. Graphical visualization of the steps that samples go through, from collection to achieving sequencing data.
Collection and preservation
Collection might seem simple, but the moment blood is drawn, tissue collected, or other samples taken, changes in cells and biological molecules start to happen, as they are exposed to a new environment.
Keerthana Krishnan, Ph.D., Director of Next Generation Sequencing Application and Product Development at New England Biolabs, emphasized that it is crucial to take good care of samples from the moment of collection: “Sample collection tubes can help to mitigate the risks posed by contaminants, as they contain preservatives that prevent cell lysis and can stabilize nucleic acids.”
Preservation is needed to maintain samples close to their biological state before prep and sequencing. Kevin Mayer, Sr. Research Scientist at Promega, explained best practices for preservation: “Good preservation comes down to three things: the right container, the right buffer, and the right temperature.”
Mayer mentioned the importance of the tube’s material (some bind nucleic acids) and airtight sealing, to stop CO2 exchange and acidification, which can lead to nucleic acid degradation. Buffers matter for pH, which can promote the hydrolysis of the nucleic acid backbones. Thaw-freeze cycles also damage nucleic acids, leading to fragmentation and damage to 3’ and 5’ sections, which can cause trouble during library prep.
“The sequencer itself is essentially a reporter and will read whatever the sample has. It has no way to distinguish a true variant from an artifact. The saying ‘garbage in, garbage out’ very much applies to NGS,” Mayer said.
All these are less important when dealing with high-quantity and quality samples, but for some applications with low amounts of collected DNA/RNA, like cancer monitoring or cfDNA detection from blood and urine samples, pre-analytical variables including collection and preservation show important changes in sequencing data quality.
Sample preparation
Sample preparation includes extraction of nucleic acids from samples and library preparation.
For most samples, manual extraction with standard reagents may work well. But some samples are more complicated. “FFPE, fresh-frozen tissue, blood, urine, and stool each present their own challenges. Urine and stool are complex matrices with inhibitors that can carry through extraction and interfere with downstream enzymatic steps,” Mayer explained.
Manual and automated kits for extraction exist to make this step easier, from the automated Promega kits such as the Maxwell CSC Blood DNA Kit or RNA Blood Kit, to the NEB manual Monarch HMW DNA Extraction Kit for Cells & Blood.
Library preparation turns a DNA/RNA sample into a group of nucleic acid fragments that can be read by the sequencer. While some view this step as a routine, one-size-fits-all step, Kristina Giorda, Ph.D., Associate Director of NGS Product Management at Watchmaker Genomics, disagreed, stating that library prep was “a critical control point that directly impacts sequencing quality, workflow scalability, and downstream biological interpretation.”
Rescuing difficult samples: library prep chemistry for the real world
Difficult samples are often the norm in clinical settings. FFPE, cfDNA, and low-input RNA are not the exception when it comes to translational research and disease diagnostics.
Let’s take a look at the latest advances allowing better analysis and sequencing of such challenging samples.
Advances in preservation for fragile samples
While new products are entering the market, basic care is still one of the most important things in sample preservation: “Sample storage in cool conditions and processing as soon as possible can help to improve outcomes,” Dr. Krishnan stated.
But for challenging samples, using the right collection tubes or adding preservation products will help. The NEB Monarch line includes the Monarch StabiLyse™ DNA/RNA Buffer, which helps preserve nucleic acid stability during sample storage and serves also as a sample lysis buffer.
Zymo Research commercializes the DNA/RNA Shield reagent, which helps reduce freeze-thaw nucleic acid damage and allows DNA/RNA preservation at room temperature from fresh samples.
When preservation is paramount, look for collection kits or similar reagents that will allow nucleic acids to remain whole before analysis. Dr. Krishnan also mentioned that “Sample preservation buffers compatible with long-term room-temperature storage and direct sample-to-library preparation methodologies are areas of active interest for our product development team,” so new products from NEB or other companies will likely become available over time.
Library prep kits and tips for low-input samples
Low input, fragmented, or damaged samples can result in “uneven coverage, increased sequencing artifacts, reduced library complexity, and challenges with downstream variant detection,” said Dr. Giorda.
Watchmaker Genomics’ DNA workflows were “engineered specifically to help address these limitations,” focusing on “maximizing library conversion efficiency while minimizing workflow-induced artifacts and preserving coverage uniformity across challenging genomic regions,” Dr. Giorda explained.
On the RNA side, the Watchmaker RNA Library Prep Kit with Polaris Depletion was designed “to support researchers working with challenging, low-input, and clinically relevant RNA samples where sensitivity, workflow efficiency, and operational scalability are especially important,” using combined rRNA and globin removal to keep more reads on target.
Other vendors have taken similar approaches for difficult, low-input samples, including cfDNA and FFPE-oriented kits from Illumina, IDT, and Takara Bio, as well as single-cell and low-input RNA-seq kits from NEB and other suppliers.
Reproducibility through automation
Variability in sequencing data can also come from human error and variability when performing manual protocols. Small changes in pipetting, cleanup, incubation timing, or normalization can affect library yield and downstream coverage. Automation helps reduce these issues, and Promega sells its Maxwell instrument and automated DNA/RNA extraction kits to help researchers reduce variability.
“Long, multi-step extractions or library prep protocols are where human inexperience, fatigue, distraction, and subtle technique variation compound across samples.” He added that “an automated platform executes the same protocol identically on sample one and sample ninety-six, at hour one and hour eight,” Mayer mentioned.
This is especially relevant in microbiome and metagenomics work, where inter-study comparisons are highly sensitive to workflow differences.
Automation is likely to improve and simplify workflows in the future. “Integrated sample-to-answer platforms—where extraction, library prep, and sequencing happen in a single closed system with minimal hands-on time—are moving from research tools toward clinical reality,” Mayer said, adding that this would reduce pre-analytical variability because it shrinks the window in which human handling can introduce error.
Beyond Promega, many labs rely on automated liquid handlers and dedicated NGS workstations from vendors such as Hamilton, Beckman, Tecan, QIAGEN, SPT Labtech, and others. Many kits from manufacturers can be run both manually and in automated platforms, making it easier for researchers to adopt automated liquid handlers.
Overall, taking care in collection, preservation, and library prep is always a good idea, but it can define whether your sequencing data is relevant or not when you work with challenging samples. Using the right combination of reagents, kits, and automation, will help minimize pre-analytical variables to obtain the best data possible.
About the author: Darío Sánchez Martín is a scientific writer and molecular biologist, specializing in biotechnology, molecular biology, and nanotechnology. He holds a Ph.D. in Biotechnology from Uppsala University, where he developed nanoparticle-based visual and magnetic assays to detect antimicrobial resistance genes. He co-founded and works at Helixa Communications as a scientific writer, helping life-science companies turn complex science into clear and persuasive content for diverse audiences.