FFPE Samples for NGS: Challenges, Quality Control & Best Practices

Formalin-fixed, paraffin-embedded (FFPE) tissue is widely used in cancer research and molecular oncology. Pathology archives provide valuable specimens for studying genetic alterations, but preparing these samples for next-generation sequencing (NGS) presents unique challenges.

The same formalin fixation process that preserves tissue morphology can also fragment and chemically modify DNA and RNA. These changes may affect extraction efficiency, library preparation, and sequencing data quality.

Understanding FFPE-related damage, optimizing sample preparation, and applying appropriate quality control measures can help laboratories improve the consistency of their NGS workflows.

Why FFPE Samples Matter for NGS

Fresh-frozen tissue generally provides better preservation of nucleic acids, but frozen specimens are not always available. FFPE blocks are routinely collected and stored in pathology workflows, making them an important resource for oncology research.

FFPE specimens can support meaningful genomic analysis when laboratories account for sample degradation, pre-analytical variability, and assay-specific input requirements.

Successful FFPE sequencing depends on more than the sequencing platform itself. Tissue selection, extraction chemistry, nucleic acid quality, and library preparation all influence the outcome.

How Formalin Fixation Affects DNA and RNA

Formalin fixation introduces chemical changes that can complicate molecular analysis. Common challenges include:

• Cross-linking: Formaldehyde-induced cross-links can make nucleic acids more difficult to recover and may interfere with amplification.
• Fragmentation: DNA and RNA may become highly fragmented, reducing the amount of usable template for certain sequencing applications.
• Base modifications: Cytosine deamination can produce artifactual C>T and complementary G>A variant calls.
• Reduced amplifiability: Chemically damaged nucleic acids may not amplify efficiently, even when measurable quantities are present.

These effects can contribute to reduced library complexity, uneven coverage, allele dropout, or false-positive variant calls.

However, the extent of damage varies between specimens. Fixation conditions, tissue characteristics, block age, storage, and extraction methods all contribute to sample quality.

Pre-Analytical Factors That Influence FFPE Quality

Many factors affecting FFPE nucleic acid quality are established before the specimen reaches the molecular laboratory.

Important considerations include:

• Fixation conditions: Neutral buffered formalin and standardized fixation procedures help reduce avoidable variability.
• Time to fixation: Delays between tissue collection and preservation can affect nucleic acid integrity.
• Fixation duration: Inadequate or prolonged fixation may compromise molecular performance.
• Storage conditions: Block age and storage environment can influence the quality of recovered material.

For archival specimens, laboratories may have limited control over the original processing conditions. Recording available specimen history can help explain differences in extraction and sequencing performance.

Selecting the Right Tissue Area

Not every region of an FFPE block provides equally suitable material for sequencing.

Before extraction, consider:

• Reviewing an H&E-stained section to identify the area of interest.
• Assessing tumor content relative to the sensitivity requirements of the planned assay.
• Avoiding highly necrotic tissue where practical.
• Using macrodissection or microdissection when enrichment is appropriate.
• Ensuring sufficient tissue is available for the required DNA and RNA inputs.

Tumor content is particularly important for detecting low-frequency variants because non-tumor material may dilute the fraction of altered sequences within a sample.

FFPE DNA and RNA Extraction Best Practices

FFPE extraction workflows must address paraffin removal, tissue digestion, nucleic acid recovery, and formalin-related chemical modifications.

Key considerations include:

• Complete deparaffinization: Follow the extraction method’s instructions to remove paraffin before purification.
• Appropriate digestion: Proteinase K digestion can help release nucleic acids from fixed tissue.
• Cross-link reversal: Use the conditions specified by the extraction chemistry rather than applying a universal heating procedure.
• FFPE-compatible reagents: Select extraction methods intended or appropriately qualified for fixed tissue.
• Suitable elution conditions: Choose elution volumes that balance nucleic acid recovery and downstream concentration requirements.
• DNA and RNA requirements: Confirm whether separate extractions or a combined workflow are needed for the planned sequencing application.

For additional guidance on purification technologies, read our Nucleic Acid Extraction for PCR and NGS guide .

Quality Control: Five Checks Before NGS Library Preparation

FFPE sample quality cannot be determined by nucleic acid quantity alone. Laboratories should evaluate whether the recovered material is suitable for the intended sequencing workflow.

Acceptance criteria should be defined for the specific assay. When a sample does not meet the required thresholds, options may include re-extraction, alternative preparation methods, or excluding unsuitable material.

Recognizing FFPE-Related Sequencing Artifacts

Formalin-induced DNA damage may introduce sequencing artifacts that resemble genuine genetic alterations.

Cytosine deamination can generate apparent C>T and G>A substitutions, sometimes at low variant allele fractions.

Depending on the workflow, laboratories may reduce the impact of artifacts through:

• FFPE-compatible library preparation and appropriate repair chemistry.
• Suitable error-correction and variant-filtering strategies.
• Reviewing variant allele fractions alongside coverage and quality metrics.
• Orthogonal confirmation of selected unexpected findings.
• Use of matched normal material when appropriate and available.

The appropriate strategy depends on the sequencing platform, library preparation method, and research objectives.

What to Look for in an FFPE-Compatible NGS Panel

When evaluating NGS panels for FFPE specimens, consider whether the assay has been designed and evaluated for the challenges associated with fixed tissue.

Important questions include:

• Is the panel compatible with FFPE tissue and limited specimens such as fine needle aspirates (FNA)?
• What are the required DNA and RNA input quantities?
• Which variant types can the assay detect?
• Does RNA fusion detection require predefined fusion partners?
• What sequencing instruments and analysis workflows are supported?
• What quality control recommendations and technical documentation are available?

For laboratories comparing sequencing with targeted amplification approaches, see our NGS vs. Real-Time PCR guide .

FFPE Precision Oncology NGS Solutions from Jant Pharmacal

Jant Pharmacal offers targeted next-generation sequencing solutions for molecular oncology research, including an integrated panel designed for FFPE and fine needle aspirate specimens.

Jant Pharmacal also offers a broader selection of NGS solutions for different research needs, including standalone RNA fusion analysis, liquid biopsy research, and hereditary genetics applications.

Explore the Jant Pharmacal NGS Portfolio .

Frequently Asked Questions About FFPE and NGS

Conclusion

FFPE tissue provides an important resource for molecular oncology research, but its successful use in NGS depends on careful sample selection, extraction, quality control, and sequencing workflow design.

Understanding formalin-related damage and evaluating both DNA and RNA quality can help laboratories reduce avoidable sequencing failures and improve the consistency of their research workflows.

Molecular research products discussed in this article are for Research Use Only (RUO) unless otherwise specified. RUO products are not intended for use in diagnostic procedures. Refer to individual product documentation for intended use.