How to Sequence a Gene: Step-by-Step Experiment Workflow

Introduction — What Does It Mean to Sequence a Gene?

Gene sequencing is the process of determining the exact order of nucleotides—adenine (A), thymine (T), cytosine (C), and guanine (G)—within a DNA molecule. In practical terms, it tells researchers the precise genetic "blueprint" that defines a gene's structure and function. Understanding this sequence enables scientists to investigate how genes function, how they vary between organisms, and how mutations impact biological processes.

In academic and contract research laboratories, gene sequencing is one of the most common workflows used to verify cloning results, identify mutations, and characterize newly discovered genes. Even though sequencing technologies have evolved dramatically—from Sanger sequencing to modern high-throughput next-generation platforms—the core experimental logic remains the same: extract, amplify, purify, and read the sequence.

Following a systematic workflow is critical to obtaining reliable and interpretable results. Poor-quality DNA, suboptimal primers, or inadequate purification can lead to unreadable chromatograms or noisy data, wasting time and reagents. By understanding each experimental step in sequence determination, laboratory staff can design better experiments and troubleshoot common issues before data analysis.

As this guide explains the step-by-step experimental workflow for sequencing a gene, it will focus on practical aspects that directly affect data quality—especially for academic researchers and CRO technicians who routinely perform gene-level sequencing. For guidance on primer design, you may also refer to our companion article, How to Design Primers for DNA Sequencing: A Practical Guide.

Step 1: Extract High-Quality DNA from Your Samples

Successful gene sequencing begins with extracting DNA that is intact, pure, and of sufficient quantity. Poor DNA quality is a frequent root cause of failed PCRs or unreadable sequencing results. Below are key practices, caveats, and tips for reliable extraction in research labs and CRO settings.

2.1. Sample Collection, Storage & Pre-treatment

• Choose fresh or well-preserved samples. Degraded tissue or prolonged freeze–thaw cycles reduce DNA integrity.
• For cells, tissues, or microbial cultures, collect at optimal growth stage (e.g., ~80% confluency for adherent cells).
• Use DNA-free, nuclease-free consumables (tips, tubes, reagents) to avoid contamination.
• Snap-freeze or store at –80 °C for long-term storage; short-term storage may use 4 °C in buffer.
• If tissues are rigid or inflexible (e.g.,, plant, fibrotic tissue), pre-homogenize (grinding, bead beating) to aid efficient lysis.
• Tissue homogenization is critical to maximizing yield.

2.2. Cell Lysis and Nuclease Inactivation

• Use a lysis buffer that contains a detergent (e.g., SDS, Triton X-100) plus a chaotropic salt (e.g., guanidine isothiocyanate) to disrupt membranes and denature proteins.
• Add Proteinase K (or other protease) during lysis to digest proteins (including histones and nucleases).
• Incubate at an optimal temperature (e.g., 55–65 °C) with occasional mixing until complete digestion is achieved.
• Include EDTA or other chelators to sequester divalent ions and inhibit DNases.

2.3. DNA Binding / Separation of Impurities

After lysis, you must separate the DNA from proteins, lipids, and other cellular debris. Common strategies include:

• Silica column / membrane binding (spin columns): DNA binds to silica under high-salt conditions, then washed and eluted.
• Magnetic bead–based capture: DNA binds beads; magnets separate beads from solution, enabling wash steps.
• Organic extraction (phenol: chloroform): Classical method, but involves hazardous solvents and is more laborious.
• Precipitation (ethanol / isopropanol + salts): Useful for concentrating DNA, but less selective and riskier for co-precipitating contaminants.

When choosing a method, consider the trade-offs: columns and beads provide cleaner DNA more quickly; organic extraction may yield more DNA from challenging samples if handled carefully.

2.4. Washing and Elution

• Wash steps (e.g., 70 % ethanol) remove salts, detergents, and residual impurities.
• It is vital to dry or spin-dry the column/bead pellet fully to remove residual ethanol (which can inhibit downstream enzymes).
• Elute DNA in a low-salt buffer (e.g., TE, Tris) or nuclease-free water. Pre-warming the elution buffer to ~50 °C can improve yield.
• Use minimal elution volume compatible with downstream steps to increase effective concentration.

2.5. Quality Control: Yield, Purity & Integrity

Before proceeding to PCR and sequencing, assess DNA quality using:

• Spectrophotometry (A260/A280, A260/A230): Preferred purity ranges ~1.8 (protein-free) and >1.8 for 260/230 (salt contaminants).
• Fluorometric quantification (e.g., Qubit) to get accurate concentration of double-stranded DNA.
• Agarose gel electrophoresis: Visualize high-molecular-weight band; detect degradation or smear.
• Optional: capillary electrophoresis or TapeStation / Bioanalyzer for more precise size/integrity.

If DNA shows degradation, or low yield or poor purity, revisit lysis conditions, wash steps, or sample handling.

Step 2: Amplify the Target Gene Using PCR

Once you have high-quality DNA, the next critical step is to selectively amplify your gene of interest by polymerase chain reaction (PCR). A well-designed, optimized PCR is foundational for clean sequencing reads later on.

3.1. Why PCR Before Sequencing?

• PCR increases the number of DNA copies for the target region to improve signal strength in sequencing.
• It enriches for the correct fragment amidst a complex genomic background.
• An overly complex sample (e.g., entire genomic DNA) without enrichment often leads to weak or ambiguous reads.

The regulatory sequencing guidelines emphasize that a robust, specific PCR is a significant determinant of sequencing success.

3.2. Reaction Components & Concentrations

A typical PCR mix includes:

3.3. Thermal Cycling Strategy

A standard PCR program often follows:

• Initial denaturation (~95 °C, 2 min) — fully denature template DNA.
• Denaturation step in each cycle (95 °C, 15–30 s)
• Annealing step (Tm – ~5 °C, ~15–30 s) — primers bind to target.
• Extension step (typically 68–72 °C, ~1 min per kb)
• Final extension (68–72 °C, 5–10 min) — ensures full-length extension.
• Hold at 4 °C.

If your target has a high GC content or secondary structures, consider touchdown PCR—starting with annealing at a higher temperature and gradually lowering it with each cycle to improve specificity.

3.4. Choosing Polymerase & Fidelity Concerns

• Use a high-fidelity polymerase (proofreading) to minimize error rates in your amplicon, especially if downstream sequencing is sensitive to mismatches.
• Some polymerases are tailored to amplify AT- or GC-rich regions. For example, Phusion Plus has been shown to amplify extremely AT-rich templates (up to 90 % AT) with proper optimization.
• When amplifying GC-rich templates, consider using master mixes with GC enhancers, or adding DMSO, betaine, or other additives to destabilize secondary structures.

3.5. Advanced PCR Strategies & Troubleshooting

• Nested PCR: useful when specificity is low. Perform a first round with outer primers, then a second round with inner (nested) primers to reduce off-target amplification.
• Hot-start PCR: prevents non-specific primer binding or extension at room temperature by delaying polymerase activity until the first denaturation step. This increases specificity.

Troubleshooting tips:

• If you see multiple bands or a smear, increase the annealing temperature or reduce the cycle number.
• If no amplification: check primer design, template concentration, or magnesium level.
• For weak bands: increase extension time or adjust polymerase concentration.

Step 3: Purify the PCR Product for Downstream Sequencing

After amplification, your PCR product still contains leftover primers, free nucleotides (dNTPs), DNA polymerase, salts, and buffer components. These contaminants can severely degrade sequencing performance—introducing noise, shortening read length, or causing reading failures. Thus, the cleanup step is essential.

Below, I describe the major purification strategies, pros/cons, and practical tips to get sequencer-ready DNA.

4.1. Why Purification Matters

• Unincorporated primers can generate spurious sequencing starts or background peaks.
• Excess dNTPs disturb the ratio between dNTP and labeled terminator nucleotides (in Sanger sequencing), which lowers signal clarity.
• Residual polymerase or buffer salts may inhibit sequencing enzymes or reduce read quality.
• When only a single clean PCR product band is present, a simpler cleanup is often sufficient; if multiple bands appear, gel purification is safer.

4.2. Common Purification Methods

Below are commonly used cleanup strategies, with comparative notes.

4.3. Practical Tips & Best Practices

• Choose method based on PCR specificity: If your PCR yields a single clean band, enzymatic or column cleanup is fastest and safest. If multiple bands appear, use gel extraction to isolate the correct fragment.
• Control elution volume vs concentration: Use minimal elution volume (e.g., 20–30 µL) to maintain sufficient concentration for sequencing.
• Dry beads or spin columns well: Residual ethanol from wash buffers can inhibit sequencing enzymes (especially polymerases).
• Pre-warm elution buffer (~50 °C) to increase DNA recovery.
• Validate purified product on gel or by spectrophotometry to ensure absence of primer dimers or smear.
• For enzymatic cleanup protocols:

    - A typical ExoI + SAP protocol from Thermo Fisher uses ~5 µL PCR product + 0.5 µL ExoI + 1 µL SAP, incubate 37 °C for 15 min, then 85 °C for 15 min to inactivate.

• Optimize bead ratio (for SPRI methods): Adjust bead : reaction volume ratio to exclude smaller fragments (e.g.,, primer dimers) while retaining full-length amplicon.
• Minimize UV exposure: During gel extraction, use minimal UV intensity or Blue Light transillumination to reduce DNA damage.

Step 4: Choose a Gene Sequencing Method

Once your PCR product is clean, the next decision is which sequencing technology is appropriate for your objective. This choice directly impacts cost, throughput, read length, accuracy, and downstream workflows. Below is a comparison of the main options and guidance on selecting the right method for gene-level sequencing.

5.1. Sanger Sequencing (Capillary / Dideoxy Method)

How it works (briefly):

• The classic dideoxy-terminator chemistry uses fluorescently labelled ddNTPs to terminate DNA strand extension at each base position.
• Fragments differing by single nucleotides are separated by capillary electrophoresis; detectors record fluorescence to infer base order.
• Typically yields read lengths up to ~800–1,000 bp (usable region ~500–800 bp).

Strengths:

• Very high base accuracy (commonly > 99.9 %)
• Straightforward data output (chromatograms with minimal computational requirements)
• Ideal for verifying single genes or small numbers of targets
• Minimal infrastructure overhead compared with high-throughput systems

Limitations:

• Low throughput (one fragment per reaction) — not cost-effective when scaling to many genes
• Read length ceiling limits use for longer amplicons or complex regions
• Sensitivity to low-abundance variants is modest (rare allele detection is difficult)

Best-use scenarios:

• Validating variant calls from high-throughput data
• Confirming plasmid inserts or cloned gene constructs
• Projects with few amplicons where setup cost must remain low

5.2. Next-Generation Sequencing (NGS / Massively Parallel Sequencing)

Overview & principle:

NGS methods sequence many DNA fragments in massively parallel fashion, enabling simultaneous reads across thousands or millions of amplicons.

Common NGS types include sequencing-by-synthesis (Illumina), ion semiconductor (Ion Torrent), and single-molecule long-read platforms (PacBio, Oxford Nanopore).

Advantages:

• High throughput: many genes or samples can be multiplexed in one run
• Deep coverage: supports sensitive detection of low-frequency variants
• Flexible scale: suitable for panel, amplicon, or even small genome sequencing
• Lower cost per base when scaling

Challenges & trade-offs:

• Short read length (for many platforms) may complicate mapping in repetitive or structurally complex regions
• Requires library preparation (not covered here)
• Data processing and bioinformatics overhead is larger
• Errors (especially systematic) must be mitigated by QC and depth

Notable case example:

In a study of foot-and-mouth disease virus, NGS uncovered low-frequency variants present at <1% in the viral population—variants that Sanger sequencing would have missed.

5.3. Long-Read / Single-Molecule Sequencing

While often thought of in genome-scale contexts, long-read platforms can sometimes be applied to gene sequencing, especially where structural variation or repetitive domains are involved.

• PacBio / SMRT sequencing: produces reads of multiple kilobases with relatively high consensus accuracy after error correction
• Oxford Nanopore: can generate very long reads, is flexible, and supports real-time basecalling

These methods help resolve difficult regions (e.g., repeats, GC-rich domains) or phasing of variants across one molecule.

Example: A microbial genome assembly study found that single-molecule long reads reduced assembly complexity and closed gaps that short reads could not resolve.

5.4. Decision Matrix: Which Method Fits Your Project?

Practical recommendations:

• If you only have one or a few genes to sequence, Sanger is reliable and cost-efficient.
• For projects with multiple genes, gene panels, or multiplexed samples, NGS offers strong scalability.
• If your gene contains highly repetitive motifs or you want to capture long-range structural variation, consider long-read sequencing.
• You may also adopt a hybrid approach: e.g., sequence with NGS broadly and validate specific variants with Sanger.

Step 5: Analyze and Validate Sequencing Data

After you receive raw sequencing data (e.g., chromatograms, FASTQ files), the next phase is to verify its quality, call the correct sequence, and confirm that the result truly represents your target gene. Poor analysis or unchecked errors can mislead your conclusions. In this section, I walk through best practices for both Sanger and NGS-based sequence validation.

6.1. Sanger Sequencing: Evaluating Electropherograms & Base Calls

For Sanger sequencing, the primary output is a chromatogram (.ab1 or similar), showing peaks of fluorescence across base positions. Key checks include:

• Peak sharpness and spacing: Ideally, well-resolved, symmetric peaks with no overlapping tails.
• Baseline noise: Minimal background signal between peaks indicates a clean read.
• Phasing and drop-off: Over time, signal may decay; quality often declines after ~700–900 bases (RTSF Genomics Core best practices).
• Quality scores / confidence calls: Many viewers display Phred-like quality scores for each base; flag lower-quality positions (e.g., < Q20).
• Ambiguous or double peaks: Mixed or overlapping peaks may reflect heterozygosity, contamination, or secondary structures.
• Directional repeat check: Always sequence from both forward and reverse primers when possible, especially in critical regions.

If base calls are ambiguous or low confidence, inspect manually and consider re-sequencing or designing alternative primers.

Guidelines from the Association for Clinical Genomic Science (ACGS) emphasize consistent criteria for calling variants, flagging uncertain bases, and reporting confidence (though their context is clinical, the principles are still relevant for rigorous research use).

Case in point: The RTSF Genomics Core published contrasting "good vs poor" chromatograms to show how incorrect template or primer concentrations degrade data.

6.2. NGS / High-Throughput Sequencing: QC, Alignment & Variant Calling

When using NGS or massively parallel sequencing, the analysis pipeline is more complex. The main stages are:

Quality control (QC) of raw reads (FASTQ)

• Trim adapter sequences, low-quality ends, and remove reads below length threshold.
• Assess base quality distributions (e.g., via FastQC or similar).
• Filter or flag reads with excessive N bases or low complexity.

Alignment / Mapping to a Reference

• Map reads to a reference gene or genome using tools like BWA, Bowtie2, or minimap2.
• Consider mismatches, indels, and mapping quality (MAPQ) scores.

Consensus Sequence / Variant Calling

• Collate aligned reads, derive a consensus base at each position (for amplicon sequencing).
• Call single-nucleotide variants (SNVs) or indels using variant callers (e.g.,, GATK, FreeBayes, DeepVariant).
• Filter variants by parameters like depth (DP), allele frequency (AF), base quality (QUAL), strand bias, and mapping score.

Validation & Cross-checking

For uncertain positions or low-frequency alleles, cross-check with Sanger sequencing (orthogonal confirmation). Many labs use Sanger to validate 1–2% of variant calls or ambiguous ones.

Note that literature debates the blanket requirement for Sanger validation; one study showed that a single round of Sanger may incorrectly refute a true-positive NGS call more often than identify false positives (i.e., false negatives) .

In a whole-genome sequencing context, a recent study on 1,756 variants found ~99.72 % concordance between WGS and Sanger when high-quality thresholds (QUAL ≥ 100, DP ≥ 20, allele frequency ≥ 0.2) were applied.

The shifting consensus is that labs should establish internal quality thresholds for which variants require orthogonal confirmation, rather than always confirming everything.

Review & Manual Curation

• Manually review variant calls in a genome viewer (e.g., IGV) especially around indels, homopolymers, or low coverage zones.
• Flag regions of low coverage or ambiguous mapping for caution or re-sequencing.

6.3. Common Pitfalls & Quality Control Tips

• Low coverage / depth: If read depth is too low, variant calls are unreliable—aim for > 20× coverage in amplicon sequencing.
• Strand bias / direction bias: If most reads supporting a variant come from one strand, that may indicate artefact.
• Variant allele frequency (VAF) threshold: For clonal genes, VAF should approach ~100 %; for mixed or heterozygous samples, expect ~50 %. Very low VAF (< 5 %) often signals noise.
• Caller inconsistencies: Use more than one variant caller or consensus filtering to reduce false positives.
• Error-prone regions: Homopolymers, GC-rich stretches, secondary structure motifs can lead to miscalls—interpret with caution.
• Batch effects & index hopping (in multiplexed NGS): Be aware of sample cross-contamination or barcode misassignment.

Practical Tips — Avoiding Common Gene Sequencing Errors

Even with a solid workflow, small mistakes or oversights can degrade your sequencing data. Here are experience-driven tips and best practices to reduce errors, boost success rates, and maintain reproducibility.

7.1. Work with Clean Techniques & Controls

• Segregate work areas for pre- and post-PCR steps, with dedicated pipettes, gloves, and consumables.
• Use filter (aerosol) tips at every pipetting step to guard against cross-contamination.
• Always include negative controls (no template) during PCR and sequencing reactions.
• For critical samples, run technical replicates or replicate sequencing to confirm consistency.

7.2. Optimize Template & Primer Concentrations

• Too much template can lead to pull-up peaks or signal saturation in Sanger chromatograms. Eurofins warns that excessive template is a known cause of distorted peaks (e.g., "very strong signals and pull-up peaks").
• Too little template often yields weak signal or unreadable traces.
• Primer concentration should be balanced—excess primer may produce primer-dimer artifacts, while too little reduces yield.
• Re-design primers if overlapping binding, secondary structure, or off-target binding appears.

7.3. Minimise PCR-Introduced Artefacts

• Prefer high-fidelity, proofreading polymerases to reduce misincorporation and indels.
• Limit cycle number to avoid over-amplification, which increases nonspecific products and error accumulation.
• Use hot-start polymerases to prevent premature extension and nonspecific amplification at room temperature.
• For GC-rich or structure-prone regions, add co-solvents (e.g., DMSO, betaine) or specialized buffers.
• Avoid "chimera formation" in multiplex or high-cycle PCRs, which can create fused or misleading amplicons.

7.4. Monitor and Limit Contaminants & Inhibitors

• Carryover from DNA extraction (e.g., salts, ethanol, phenol) can inhibit polymerases—make sure wash and elution steps are thorough.
• If inhibition is suspected, dilute your template or re-purify it (e.g., ethanol precipitation or clean-up kit).
• Use freshly prepared reagents, avoid repeated freeze–thaw cycles, and discard old or suspect stocks.
• Use ultrapure nuclease-free water and check for contaminants (e.g., nucleases, nucleotides) in buffers or stock reagents.

7.5. Choose the Right Sequencing Primer & Strategy

• The primer used in sequencing ("cycle sequencing primer") may differ from PCR primers—some PCR primers perform poorly in linear sequencing reactions.
• In homopolymer or repetitive regions, consider anchored primers, which "lock in" binding across repeats and reduce slippage artifacts.
• Sequence from both forward and reverse directions when possible, especially over difficult motifs or ambiguous bases.

7.6. Inspect Chromatograms & Early Quality Checks

• Use software (e.g., Sequence Scanner, TraceViewer) to assess signal-to-noise ratio, peak separation, baseline drift, and dye blobs.
• Watch for "mixed peaks," uneven spacing, or signal drop-off—these may indicate contamination, primer issues, or mis-priming.
• If a read fails or is ambiguous in one direction, re-sequence with a new primer or adjust template amount.

7.7. Document and Log Every Condition

• Always record batch numbers (kit lot, polymerase, reagent lot) along with exact conditions (temperatures, times, concentrations).
• Include metadata: sample source, extraction date, storage conditions.
• Over time, build a lab-specific error log—you may detect trends (e.g., particular batches giving low yields).

Tools and Reagents Checklist

Below is a consolidated checklist of essential tools, reagents, and consumables for performing gene sequencing via PCR + downstream sequencing. Use this as a quick lab reference and as part of your internal SOP documentation.

Conclusion

Sequencing a gene is a stepwise journey—from starting material to a clean, validated sequence. By methodically following the workflow (DNA extraction → PCR amplification → purification → sequencing → analysis) and applying the practical tips outlined above, you maximise success and reproducibility in your experiments.

In research labs and CROs, small errors (contamination, suboptimal primers, incomplete cleanup) often derail sequencing results. But by enforcing good lab practices, rigorously checking DNA quality, and choosing the right sequencing method (Sanger, short-read NGS, or long-read), you can reduce failure rates and improve data reliability.

If your project involves multiple genes, panel sequencing, or requires deeper variant sensitivity or structural insight, you might consider outsourcing parts or all of your workflow to a specialized genomics service provider. For example, at CD Genomics, we support full sequencing services—from sample QC, to sequencing, through bioinformatics delivery—tailored for academic, industrial, and pharmaceutical clients.

Next steps (Action):

If you'd prefer to focus on your science and leave sequencing execution to experts, contact us for a consultation or request a quote for your project.

•  To deepen your understanding, explore these related articles in our content library:
• How to Design Primers for DNA Sequencing: A Practical Guide (primer strategies)
• Sample Preparation for High-Quality Sequencing Results (DNA extraction best practices)
• Library Preparation Strategies for Next Generation Sequencing (for scaling to NGS workflows)

Let us help you move from sample to sequence with confidence.

Frequently Asked Questions (FAQs)

Q: How do you sequence a gene from start to finish?

To sequence a gene, you first extract high-quality DNA from your sample, then amplify your target region via PCR, clean up the PCR product to remove residual primers and dNTPs, choose an appropriate sequencing method (e.g. Sanger or NGS), send the purified amplicon into sequencing, and finally validate the output by analyzing chromatograms or base calls and confirming with reference alignment or orthogonal methods.

Q: What factors most affect sequencing success?

Key determinants include DNA quality (purity, integrity, absence of inhibitors), primer specificity and design, PCR efficiency (correct reagents, cycles and enzyme), thorough purification of the amplicon, and appropriate sequencing depth or read quality; when any of those steps is weak, the final sequence can be noisy, ambiguous, or fail entirely.

Q: Can PCR products be sequenced directly without further steps?

Only if the PCR yield is extremely clean (single‐band with no primer dimers or nonspecific products). In most real-world experiments cleanup is essential. Unremoved primers, dNTPs, or polymerase carryover can interfere with sequencing chemistry and degrade read quality, so purification before sequencing is generally mandatory.

Q: How to choose between Sanger sequencing and NGS for a gene?

Use Sanger when you have one or a few amplicons and need very high accuracy with low throughput. Choose NGS when many genes, many samples, or multiplexed panels are required—NGS offers scalability and depth, at the cost of more complex data and library prep.

Q: What is primer walking and when is it used?

Primer walking is a method in which sequential primers are designed to "walk along" the DNA template to cover a longer region than a single read can reach; after initial sequencing, new primers are designed adjacent to the last known base, and the next segment is sequenced, continuing iteratively until the full stretch is covered (Wikipedia: Primer walking).

Q: How do I know if my sequencing result is reliable?

Check quality metrics such as sharp peaks, minimal noise in chromatograms for Sanger, or read depth, base quality scores, and mapping consistency in NGS. Also compare your sequence against a trusted reference or database and optionally confirm ambiguous or novel variants using a second method or replicate sequencing.

Q: What is the difference between whole-gene sequencing and panel or exome sequencing?

Whole-gene sequencing focuses on a narrow region (the gene of interest), using targeted PCR or capture, whereas panel sequencing covers a group of genes, and exome sequencing spans all coding regions across many genes. The deeper focus in gene sequencing usually yields higher coverage and simpler analysis pipelines.

Q: What should I do if a region fails to sequence or shows ambiguous calls?

You can redesign primers (especially internal primers), split the amplicon into overlapping fragments, reduce secondary structure via additives (e.g. DMSO), or apply alternative sequencing strategies (e.g. long reads). Re-sequencing from both forward and reverse directions often helps resolve ambiguity.

Reference:

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2. Henriette O'Geen, Marketa Tomkova, Jacquelyn A Combs, Emma K Tilley, David J Segal, Determinants of heritable gene silencing for KRAB-dCas9 + DNMT3 and Ezh2-dCas9 + DNMT3 hit-and-run epigenome editing, Nucleic Acids Research, Volume 50, Issue 6, 8 April 2022, Pages 3239–3253
3. 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.
4. Kopernik, A., Sayganova, M., Zobkova, G. et al. Sanger validation of WGS variants. Sci Rep 15, 3621 (2025).