Sample Preparation for High-Quality Sequencing Results

Why Sample Quality Determines Sequencing Success

In the world of next-generation sequencing (NGS), the quality of your input sample is often the single most significant determinant of success—or failure. Even with the most advanced sequencer and library prep kit, degraded, impure, or low-yield DNA/RNA can derail an entire run.

Key reasons sample quality matters

1. Enzymatic efficiency depends on purity.

Library preparation steps—end repair, adapter ligation, and PCR amplification—are enzyme-driven. Contaminants such as phenol, salts, or residual ethanol inhibit these enzymes.

2. Fragment integrity affects sequencing yield.

Highly fragmented or nicked DNA leads to inefficient cluster generation or poorer read mapping. In tissue-derived samples, lower DNA integrity is associated with significantly lower success rates. (Kuwata et al.; NCI SCRUM-Japan data)

3. Quantification errors propagate downstream.

Inaccurate measurement of DNA concentration leads to underloading or overloading the sequencer. Overloading reduces cluster quality; underloading wastes capacity. Fluorometric assays (Qubit, PicoGreen) are preferred over spectrophotometry for precise quantification of nucleic acids.

4. Low input increases sensitivity to contamination.

In samples with scant DNA, even trace amounts of exogenous DNA (e.g.,,, from reagents or the environment) can skew the results. Studies of unmapped reads have shown that dilute samples are particularly susceptible to contamination artefacts. (Lusk, 2014)

Impact: integrity predicts success

In one extensive analysis of formalin-fixed, paraffin-embedded (FFPE) tissues (n = 2,573), samples with high DNA integrity (ΔCt < 4.4 by qPCR metric) yielded NGS success rates of ~94%. In contrast, low-integrity samples had success rates of ~5.6%. (Kuwata et al.)

This result underscores a central point: no downstream "rescue" can fully compensate for poor starting material. The moment you compromise sample quality, you reduce your margin for error in every subsequent step.

Essential Steps in Sequencing Sample Preparation

To reliably convert raw biological material into sequencing-ready nucleic acids, labs must follow a disciplined, stepwise workflow. Below is a refined roadmap, along with caveats and best practices.

2.1 Sample Collection & Stabilization

• Choose collection tubes or preservatives that inhibit nucleases or microbial growth (e.g.,, EDTA for blood, RNAlater for RNA).
• Minimize the number of freeze–thaw cycles—each cycle degrades nucleic acids incrementally.
• Record sample metadata (source, time, temperature history) to flag anomalous variability later.

Why it matters: Poor handling at this stage amplifies downstream artifacts like fragmentation, contamination, or loss.

2.2 DNA / RNA Extraction

This core step isolates nucleic acids from cells, tissues, or fluids. The method choice (column-based, magnetic beads, phenol/chloroform, or automated systems) depends on sample type, throughput, and purity requirements.

Key considerations & best practices:

• Use nuclease-free, certified reagents and consumables (tips, tubes, pipette filter tips).
• After lysis, ensure complete removal of proteins, salts, detergents, phenol, and residual ethanol. Illumina warns that residual inhibitors (e.g.,, phenol, EDTA, humic acids) can interfere with end repair, ligation or PCR.
• For some challenging matrices (e.g.,, plant, soil, FFPE), consider an extra cleanup or purification step (e.g.,, spin-column repurification) to remove inhibitors.
• Choose an elution buffer compatible with downstream steps (e.g.,, 10 mM Tris, pH 7.5–8.5). Illumina suggests avoiding high EDTA or acidic buffers that may reduce enzyme activity.
• For high-throughput workflows, magnetic bead–based automated platforms (e.g.,, Thermo Fisher KingFisher systems) provide consistency and reduced hands-on error.

2.3 Quantification & Quality Control (QC)

After extraction, validate your nucleic acid intensity, purity, and integrity before proceeding.

Critical checks to perform:

• Purity ratios (A260/280, A260/230): Basic UV spectrophotometry reveals contamination with proteins, phenol, or salt. Acceptable ranges: A260/280 ~1.8 for DNA, ~2.0 for RNA; A260/230 ideally > 1.8. Illumina recommends these as a first screen.
• Fluorometric quantification (Qubit, PicoGreen): More accurate for double-stranded nucleic acid quantification. Illumina's DNA Prep guide explicitly cautions against relying solely on UV methods.
• Integrity/fragmentation assessment:
• – Gel electrophoresis or TapeStation / Bioanalyzer traces (for DNA)
• – RNA Integrity Number (RIN) or equivalent metrics for RNA
• Optional QC assays:
• – Small "test" PCR amplification to detect inhibitors
• – Spike-in control or synthetic standard to monitor yield bias
• Document all QC metrics in your lab notebook or LIMS system to help diagnose failures later.

2.4 Storage & Transport of Nucleic Acid Samples

Preserving sample integrity post-extraction is as important as the extraction itself.

Guidelines for storage and shipment:

• Short-term: Keep aliquots at 4 °C if processing within hours; otherwise, –20 °C.
• Long-term: Store at –80 °C (especially for RNA).
• Buffer: Use TE buffer (10 mM Tris, 1 mM EDTA) at pH 7.5 for DNA; avoid divalent cations or buffers that promote degradation.
• Avoid repeated freeze–thaw: Aliquot samples into smaller volumes.
• Cold chain during shipment: Use dry ice or validated transport boxes to maintain temperature. Log temperature with data loggers.
• Labeling & metadata: Include identifiers, concentration, QC metrics, and handling history.

How to Improve DNA Quality for NGS

Even a well-designed extraction protocol can yield suboptimal DNA if minor details are neglected. Below are refined tactics and best practices to push your sample quality near ideal levels for sequencing.

3.1 Use High-Integrity Starting Material

• Whenever possible, begin with freshly collected or flash-frozen tissue. Repeated freeze–thaw degrades DNA over time.
• For challenging specimens (e.g.,,, FFPE, old archival samples), consider targeting shorter amplicons or using hybrid capture instead of whole-genome sequencing.
• In comparative studies, extraction methods exhibit a≥10% variation in yield and fragment size, depending on the cell type and lysis protocol (ScienceDirect, "Assessment of DNA quality" article).

3.2 Optimize Lysis and Binding Conditions

• Tailor buffer composition (e.g.,, using sufficient detergents, proteinase K, and optimal salt concentrations) to fully break down cell walls, membranes, and protein complexes.
• For samples with high polysaccharides or phenolic content (e.g.,, plants, soil), include additional cleanup steps or binding buffer additives (e.g.,, PVPP, CTAB, or inhibitor removal columns).
• Use gentle mixing (e.g.,, slow rotation) rather than vortexing to avoid mechanical shearing of genomic DNA.
• Keep incubation times just long enough for complete lysis; overincubation with harsh conditions can nick DNA ends.

3.3 Avoiding Inhibitors & Carryover Contaminants

• After the binding and wash steps, carefully remove residual ethanol—do not overdry the pellet, as this can make re-dissolution inefficient.
• Use an extra wash (e.g.,, 70 % ethanol) or a "wash buffer + 80 % ethanol" step when working with sticky contaminants.
• Include an RNAse or DNase (depending on the target) cleanup step, where necessary, to remove unwanted nucleic acid species.
• Use a final spin or vacuum step to remove residual salts or reagents (e.g.,,, guanidine, EDTA) that may inhibit downstream enzymes.

3.4 Gentle Elution Strategies

• Warm the elution buffer (e.g.,, 37 °C) prior to applying to the column or beads; let it incubate on the matrix for 2–3 minutes before centrifuging.
• Elute in low-salt buffer (e.g.,, 10 mM Tris, pH 7.5) or nuclease-free water (if acceptable) rather than buffers containing too much EDTA.
• Use elution volumes that are just sufficient for concentration without sacrificing yield. For some users, two sequential small-volume elutions yield better concentration and recovery.

3.5 Use Automation and Clean Consumables

• Automated platforms (e.g.,, magnetic bead robots) reduce human error and variation across batches.
• Always use filter-tip pipettes, sterile consumables, and dedicated pre- and post-PCR areas to ensure the highest level of safety and accuracy.
• Maintain reagent stocks properly (avoid repeated freeze–thaws) and regularly audit them for optimal performance.

3.6 Post-Extraction Polishing (Optional but Critical for Tough Samples)

• For low-purity extracts, apply a cleanup kit (e.g.,,, SPRI beads, silica columns) to further remove inhibitors.
• If necessary, perform size selection to remove very short fragments (e.g.,,, <200 bp) that may dominate during library preparation.
• Use DNA repair or end-polishing enzymes to mend nicks, but only when the starting DNA is reasonably intact.

Common Contamination Sources and How to Prevent Them

Even when every molecular step is technically sound, contamination can undo your sequencing run. Here we break down frequent contamination routes and practical prevention strategies, presented as a lab checklist framework.

4.1 Types of Contamination in Sequencing Workflows

4.2 Prevention Strategies: A Checklist Approach

Here's a practical checklist you can adopt in your lab to reduce contamination risk:

• ☐ Fully separate pre-PCR (or pre-library) and post-PCR/library areas. Never bring materials back upstream.
• ☐ Use dedicated equipment and consumables in each zone (pipettes, tubes, filter tips).
• ☐ Aliquot reagents (e.g., primers, buffers) into single-use vials to minimize repeated opening.
• ☐ Use aerosol-resistant filter tips or positive displacement pipettes.
• ☐ Decontaminate surfaces and tools regularly: bleach (10–15 %), UV irradiation, and DNA decontamination reagents.
• ☐ Include negative controls and extraction blanks in every workflow run.
• ☐ Track lot numbers of reagent kits and perform background checks or "blank runs" when switching lots. (Reagent kit variability ("kitome") has been shown to differ across batches)
• ☐ Restrict personnel movement — change gloves, lab coats, or don protective gear when crossing zones.
• ☐ Minimize PCR cycles, especially for highly amplified target regions, to reduce carryover sensitivity
• ☐ Adopt dUTP + uracil-DNA glycosylase (UNG) carry-over prevention in library or PCR prep to degrade amplicon contaminants.

4.3 Example Method: UNG + dUTP for Carry-Over Control

To limit amplification carry-over contamination, some labs incorporate dUTP in PCR products and employ uracil-DNA glycosylase (UNG) before subsequent reactions. UNG cleaves uracil bases (in prior amplicons), rendering contaminant DNA non-amplifiable while sparing native DNA templates without uracil.

One published protocol adapted this for two-step PCR library prep. They showed a significant reduction in carry-over contamination while maintaining library yield and diversity.

4.4 Note: Kit-Based Contamination in Metagenomic Workflows

A recent study examined multiple DNA extraction reagent brands and found distinct "background microbiota" signatures unique to each kit batch. Some reagents harboured microbial DNA that could bias metagenomic profiling if not controlled.

This underscores the importance of using reagent blanks as internal controls and interpreting low-abundance reads with caution.

Practical Checklist for Sample Quality Control

Below is a robust, lab-friendly checklist you can adopt to validate your DNA/RNA quality before investing in library prep. Use this as a gatekeeper to catch poor input materials early.

⚙️Sample QC Checklist: Key Metrics & Thresholds

*Thresholds may vary depending on your library protocol and input amounts; always consult your kit's documentation.

Implementation Notes & Best Practices

• Document every QC metric in your LIMS or lab notebook. Capture raw traces (e.g., gel, electropherogram) as file attachments.
• Apply gates early. If a sample fails A260/230 or fluorometric checks, do not waste reagents attempting library prep.
• Use replicate QC for critical samples. Especially for precious input material, run duplicates of quantification or integrity assays.
• Perform reagent blanks in parallel. Process "no DNA/RNA" controls to detect kit contamination or background interference.
• Flag borderline samples. Assign them to a "monitor" category — proceed with caution or additional cleanup steps.
• QC again after the cleanup or polishing steps. If you perform additional bead cleanups or size selection, repeat quantification and integrity checks.

Link to Related Content

For more on post-QC library validation and metrics, see our article "Quality Control Before Sequencing: Ensuring Data Integrity", where we dive deeper into metrics like Q30, cluster density, and read quality.

Also, this article ties in with "Library Preparation Strategies for Next Generation Sequencing", which describes how QC gates feed into efficient library workflows.

Case Study: How Rigorous Sample Prep Rescued NGS Success in a CRO-Style Workflow

In a recent institutional analysis of rare tumor sequencing, about 14.7 % of sequencing runs failed because the input material fell short in quantity or quality. (Itkin et al., 2025. DOI: https://doi.org/10.3892/mi.2025.226) Of the eight failed assays that were retested, seven succeeded after re-extraction or adjustments to prep.

Lessons for CRO Environments

From this experience, lab teams and project managers can adopt these strategies:

In practice, applying these lessons in your CROs environment could reduce failure rates from ~10 % to under 3 %.

Figure 1 - Flow diagram of the study protocol. NGS, next-generation sequencing.

Next Steps to Improve Your Sequencing Workflow

To deepen your understanding and seamlessly integrate sample prep into your broader sequencing pipeline, here are three highly relevant resources:

How to Design Primers for DNA Sequencing: A Practical Guide — Learn how primer design impacts downstream success and help avoid amplification bias.

How to Sequence a Gene: Step-by-Step Experiment Workflow — Explore the full end-to-end protocol, placing sample prep in its broader context.

Library Preparation Strategies for Next Generation Sequencing — Dive into the methods for adapter ligation, size selection, and other library construction details.

By linking your QC and prep choices to these adjacent topics, you maintain a cohesive content hub that guides readers through every stage of sequencing.

Final Takeaway

High-quality sequencing outcomes begin long before the sequencer—at sample prep. When you implement strict QC gates, contamination control, and backup strategies, you dramatically increase your success window.

If you'd like to:

• Validate your existing prep process
• Develop custom QC thresholds for your lab
• Secure a pilot sequencing project with guaranteed performance

…our expert sequencing team is ready to collaborate. Contact us today to review your protocol or request a consultation.

FAQs

Q: What causes sequencing to fail?

Sequencing failures often stem from poor sample quality — low DNA integrity, residual contaminants (phenol, ethanol, salts), or inhibitors from the extraction process can block enzymatic steps like ligation or PCR. Occasionally, cross-contamination or reagent carryover can override good input and cause complete failure.

Q: How can I improve DNA quality for NGS?

You can improve DNA quality by using fresh or properly stored starting material, optimizing lysis conditions to avoid shearing, performing extra cleanup steps to remove inhibitors, eluting gently in low-salt buffer, and using automated extraction systems with consistent performance.

Q: What are the key metrics I should check before proceeding with library prep?

You should verify purity ratios (A260/280 and A260/230), accurate concentration by fluorescence (Qubit or PicoGreen), and integrity (gel, TapeStation, or Bioanalyzer). A negative blank control helps detect background contamination before committing to library prep.

Q: How do I prevent contamination in my sequencing workflow?

Prevent contamination through physical separation of pre- and post-PCR areas, use of filter tips and dedicated instruments, decontamination (bleach, UV), reagent blanks, aliquoting reagents, restricting personnel movement, and optionally using dUTP/UNG systems to eliminate carryover amplicons.

Q: Can I rescue low-quality DNA samples?

Sometimes. If DNA is moderately impure or mildly degraded, applying polishing steps (e.g., SPRI bead cleanup, size selection, repairing nicks) may raise it above library prep thresholds. However, severely fragmented or heavily contaminated DNA often cannot be fully rescued without re-extracting.

Q: Why is quantification by UV (Nanodrop) often unreliable?

UV spectrophotometry measures all nucleic acid and absorbing substances—including free nucleotides, primers, and contaminants—leading to overestimation. Fluorometric methods (e.g., Qubit) bind specifically to double-stranded DNA and are more accurate for library prep.

References:

1. Lai Z, Su Y, Lin H, Wang S, Lin Y, Liang S, Chen W, Hsueh P. 2025. Deciphering the impact of contaminating microbiota in DNA extraction reagents on metagenomic next-generation sequencing workflows. Microbiol Spectr 13:e03119-24.
2. Jansson L, Aili Fagerholm S, Börkén E, Hedén Gynnå A, Sidstedt M, Forsberg C, Ansell R, Hedman J, Tillmar A. Assessment of DNA quality for whole genome library preparation. Anal Biochem. 2024 Dec;695:115636. doi: 10.1016/j.ab.2024.115636. Epub 2024 Aug 5. PMID: 39111682.
3. Ye, J., Coulouris, G., Zaretskaya, I. et al. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134 (2012).
4. Wright CF, Morelli MJ, Thébaud G, Knowles NJ, Herzyk P, Paton DJ, Haydon DT, King DP. Beyond the consensus: dissecting within-host viral population diversity of foot-and-mouth disease virus by using next-generation genome sequencing. J Virol. 2011 Mar;85(5):2266-75. doi: 10.1128/JVI.01396-10. Epub 2010 Dec 15. PMID: 21159860; PMCID: PMC3067773.
5. Kopernik, A., Sayganova, M., Zobkova, G. et al. Sanger validation of WGS variants. Sci Rep 15, 3621 (2025).
6. Lusk RW. Diverse and widespread contamination evident in the unmapped depths of high throughput sequencing data. PLoS One. 2014 Oct 29;9(10):e110808. doi: 10.1371/journal.pone.0110808. PMID: 25354084; PMCID: PMC4213012.
7. Kuwata T, Wakabayashi M, Hatanaka Y, Morii E, Oda Y, Taguchi K, Noguchi M, Ishikawa Y, Nakajima T, Sekine S, Nomura S, Okamoto W, Fujii S, Yoshino T; SCRUM-Japan GI-SCREEN Pathology Group. Impact of DNA integrity on the success rate of tissue-based next-generation sequencing: Lessons from nationwide cancer genome screening project SCRUM-Japan GI-SCREEN. Pathol Int. 2020 Dec;70(12):932-942. doi: 10.1111/pin.13029. Epub 2020 Oct 8. PMID: 33030786; PMCID: PMC7820973.