FFPE Spatial ATAC-seq Sample Preparation & QC Guide

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Working with FFPE tissue can be both an opportunity and a challenge. These samples preserve valuable material from past experiments and archives, yet their fixation process complicates downstream sequencing. In spatial ATAC-seq, where accurate chromatin accessibility maps depend on high-quality preparation, how FFPE material is handled becomes critical.

This article analyzes the essentials of preparing FFPE samples for spatial ATAC-seq, focusing on practical steps and quality control checks that help ensure reliable results. Whether starting with new sections or archived blocks, the following best practices will help you avoid common pitfalls and maximize your FFPE collections.

Spatial-FFPE-ATAC-seq design and data quality. (Guo, Pengfei, et al., Nature Communications 2025)

Why Do FFPE Samples Pose Challenges for Spatial ATAC-seq

Chemical crosslinking complicates chromatin access.

Formalin fixation stabilizes proteins and nucleic acids by forming strong crosslinks. While this preserves tissue architecture, it also traps chromatin in a rigid state. For assays like ATAC-seq that rely on enzyme access to open chromatin, these crosslinks become a major obstacle. The transposase cannot efficiently reach target sites, lowering the overall signal and reducing sensitivity in detecting regulatory regions.

DNA degradation during storage

Beyond crosslinking, FFPE processing often leads to fragmentation and chemical modification of DNA. Heat, pH changes, and embedding procedures can all contribute to strand breaks and base alterations. When sequenced, these damaged molecules yield short, uneven fragments and increase duplication rates, making it harder to build a complex and representative library.

Consequences for spatial profiling

In spatial ATAC-seq, degraded DNA and crosslinked chromatin reduce data yield and distort the spatial signal. Instead of clean enrichment around transcription start sites (TSSs) and apparent regional differences, researchers may see weaker peaks and noisier distributions. This means subtle differences in chromatin accessibility between neighboring tissue regions might be lost or underestimated.

Why researchers should still care

Despite these issues, FFPE remains one of the richest sources of biological material, especially for tissues that cannot be collected again. Large biobanks and clinical archives contain decades of FFPE blocks with unique research value. Optimized workflows and careful quality checks can reduce many of the barriers caused by fixation, allowing scientists to unlock valuable spatial epigenomic information from archived samples.

How Can FFPE Sample Processing Be Optimized for Spatial ATAC-seq

Preparing FFPE tissue for spatial ATAC-seq is less about following a rigid recipe and more about finding the right balance between recovery and preservation. The goal is to undo the damage caused by fixation just enough to recover usable chromatin while maintaining the tissue's architecture for spatial mapping. Below are practical strategies that researchers have found effective.

Integration of RNA-seq data with spatial-FFPE-ATAC-seq. (Guo, Pengfei, et al., Nature Communications 2025)

1. Controlled heat for crosslink reversal

Heat-induced retrieval remains the cornerstone of FFPE optimization. Applying moderate heat in an alkaline buffer can loosen formaldehyde crosslinks without destroying DNA. The art here lies in controlling both time and temperature: too little, and crosslinks persist; too much, and DNA fragments further. Researchers often test retrieval conditions in pilot sections before committing to large runs.

2. Protease treatment for protein release

Crosslinked proteins can block the transposase enzyme. A carefully timed protease step, usually with Proteinase K, helps digest these barriers. The trick is to use just enough digestion to expose chromatin while avoiding over-digestion that can compromise tissue morphology. In spatial assays, where section integrity matters, this balance is even more delicate than in bulk sequencing.

3. Buffer chemistry as a hidden variable

Not all buffers are equal. Slight differences in pH or additives can shift outcomes dramatically. Alkaline buffers (around pH 9.0) tend to improve accessibility, and including EDTA protects ion-driven DNA nicking during heating. Small tweaks here can often make the difference between a noisy dataset and a usable chromatin profile.

4. Ensuring reliable spatial barcoding

Once chromatin is made accessible, the section must accept spatial barcodes evenly. FFPE sections are prone to uneven hydration, leading to patchy labeling. Researchers have noted that fully rehydrating and gently flattening sections before barcoding helps improve barcode penetration and minimizes spatial dropout. This step is often overlooked, yet it directly affects the clarity of spatial maps.

5. Signs of success

When these optimizations are applied, FFPE spatial ATAC-seq can approach the performance of fresh-frozen samples. Reasonable mapping rates, solid TSS enrichment, and consistent fragment counts are achievable. As importantly, accessibility signals should align with known histological features, confirming that spatial fidelity has been preserved.

QC Metrics & Practical Insights from the Bench

Quality control checks whether your FFPE spatial ATAC-seq workflow is truly working. Because FFPE samples are unpredictable, relying only on standard sequencing outputs can be misleading. Instead, researchers should track a few key QC signals that reflect library quality and the integrity of spatial information.

Spatial profiling of chromatin accessibility in FFPE human thymus. (Guo, Pengfei, et al., Nature Communications 2025)

1. Duplication rate as a quick reality check

One of the first metrics people look at is the duplication rate. High duplication means the library has low complexity, often due to DNA damage or over-amplification during PCR. In published spatial FFPE datasets, duplication rates around 15–20% have been reported, which is a reasonable range for damaged samples. If your rate is much higher, it usually signals over-amplification or poor DNA recovery. My advice: adjust PCR cycles conservatively and, if possible, increase tissue input rather than "rescuing" low-yield libraries with more PCR.

2. Fragment length distribution tells a story

FFPE samples tend to skew toward shorter DNA fragments. That's expected, but the key is whether you still see the characteristic nucleosome pattern: nucleosome-free fragments (<100 bp) and mono- or di-nucleosome peaks. If that pattern disappears completely, it may indicate over-fragmentation during sample recovery or excessive DNA degradation. Keeping an eye on this distribution early can save wasted sequencing runs.

3. TSS enrichment remains a sensitive indicator

Even in FFPE material, transcription start site enrichment should be visible if the assay works. A clear enrichment peak shows that open chromatin is being captured in the right places. Weak or flat enrichment curves suggest either that crosslinks were not fully reversed or that the transposase couldn't access DNA efficiently. If this happens, re-examining your crosslink reversal step is usually more effective than adjusting sequencing depth.

4. Mapping rate reflects both sample and prep quality

A reasonable mapping rate (above ~80%) indicates that most fragments align to the genome instead of being junk reads. Lower mapping rates often come from chemical modifications or contaminating molecules introduced during FFPE processing. Simple things like verifying buffer quality, checking for residual paraffin, and ensuring proper deparaffinization can make a big difference here.

5. Don't skip spatial visualization

QC is not only numbers. For spatial assays, one of the best checks is visual: overlay fragment counts or accessibility scores onto the histology image. Do the peaks match known tissue boundaries? Are regions with dense cells showing higher signal than sparse areas? If the spatial map looks patchy or random, the problem could be uneven barcoding or incomplete rehydration of the section. This step is often the fastest way to catch issues that metrics alone can't explain.

Common Failure Modes & Mitigation Strategies

Even with careful planning, FFPE spatial ATAC-seq can fail in subtle ways. Knowing the common weak points helps you troubleshoot quickly and avoid wasting valuable archived tissue. Below are problems researchers frequently report, along with practical strategies to get experiments back on track.

Integration of RNA-seq data with spatial-FFPE-ATAC-seq from human cerebellum.( (Guo, Pengfei, et al., Nature Communications 2025)

1. Incomplete crosslink reversal

• The issue: If crosslinks are not sufficiently broken, the transposase cannot reach the DNA efficiently. This often shows up as weak TSS enrichment and a low fraction of usable fragments.
• How to fix: Extend the heat retrieval step slightly or adjust the buffer pH. Adding a mild protease digestion often improves accessibility without harming morphology. In practice, testing different retrieval times on pilot slides pays off more than pushing every sample through the same settings.

2. Degraded or heavily fragmented DNA

• The issue: Long-stored FFPE blocks sometimes produce DNA that is too fragmented for reliable library prep. The result is poor library complexity, high duplication, and noisy fragment profiles.
• How to fix: Start by checking DNA quality before sequencing. If degradation is unavoidable, consider pooling adjacent sections to increase input material. Reducing PCR cycles can also help maintain complexity instead of amplifying noise.

3. Low library complexity and high duplication rates

• The issue: Over-amplification during PCR or insufficient input DNA leads to libraries dominated by duplicates. Data may look abundant, but much of it is redundant.
• How to fix: Optimize PCR by stopping amplification as soon as usable library amounts are achieved. If tissue is limited, replicate libraries from parallel sections can improve complexity more reliably than pushing a single low-input library too far.

4. Uneven spatial signal or patchy barcoding

• The issue: If sections are not fully rehydrated or mounted evenly, barcodes may penetrate irregularly. This produces patchy or inconsistent spatial maps that don't match histology.
• How to fix: Make sure tissue sections are flat, free of residual paraffin, and hydrated properly before barcoding. A quick visual inspection under the microscope after deparaffinization can help catch problems before committing to sequencing.

5. Misinterpretation of QC metrics

• The issue: Sometimes datasets appear "failed" because researchers rely on a single QC metric. For example, short fragments alone do not always mean the library is unusable, since FFPE naturally yields smaller fragments.
• How to fix: Always interpret QC as a set of indicators—fragment profile, TSS enrichment, duplication, mapping rate, and spatial visualization together. A library can be acceptable even if one metric is imperfect, as long as the overall profile supports reliable biological interpretation.

Conclusion

FFPE tissue sits at the intersection of opportunity and challenge. On one hand, it offers access to rare and irreplaceable samples preserved over the years. On the other hand, fixation and storage introduce barriers that can easily compromise chromatin accessibility assays. For spatial ATAC-seq, where the entire point is to map open chromatin across intact tissue architecture, these obstacles cannot be ignored.

What becomes clear from both published studies and practical lab experience is that sample quality sets the ceiling for downstream analysis. Even the best computational pipeline cannot rescue poorly prepared input. By carefully reversing crosslinks, protecting DNA integrity, and monitoring key QC metrics, researchers can recover robust accessibility signals that still respect the spatial context of the tissue.

In the end, the message is simple: treat FFPE material with the respect it deserves. Optimized preparation and thorough QC turn what might look like degraded blocks of wax into high-value sources of epigenomic insight. This difference can decide whether an experiment yields a handful of noisy reads or a dataset capable of answering meaningful biological questions for research teams working with archived collections.

Supporting Researchers with FFPE Spatial ATAC-seq

Working with FFPE samples is never a one-size-fits-all process. Each block can respond differently depending on its history, fixation conditions, and storage. This is why many research teams find value in partnering with specialists who have tested multiple workflows and learned where small adjustments make the biggest difference.

At CD Genomics, we support research groups as they navigate these challenges. We provide:

• Guidance on optimizing section preparation and crosslink reversal.
• Quality control checks tailored to the quirks of FFPE material.
• Consultation on spatial barcoding and library construction to reduce wasted effort.
• Data delivery in formats that are directly usable for downstream analysis.

Our services are strictly for research applications—we do not provide clinical or personal testing. By focusing on reproducibility and reliability, we aim to help scientists turn FFPE archives into data that drives discovery.

References

1. Guo, Pengfei, et al. "Spatial profiling of chromatin accessibility in formalin-fixed paraffin-embedded tissues." Nature Communications 16.1 (2025): 5945.
2. Carraro, Caterina, et al. "Chromatin accessibility profiling of targeted cell populations with laser capture microdissection coupled to ATAC-seq." Cell reports methods 3.10 (2023).
3. Farzad, Negin, et al. "Spatially resolved epigenome sequencing via Tn5 transposition and deterministic DNA barcoding in tissue." Nature Protocols 19.11 (2024): 3389-3425.
4. Duenas, Daniela, et al. "1212" Single Cell and Spatial Analysis of Tumor Microenvironment of Nodal T Follicular Helper Cell Lymphoma, Angioimmunoblastic Type: Understanding under CO-detection by Indexing (CODEX)"." Laboratory Investigation 105.3 (2025).