Cancer research depends on the details we can actually see. And, until now, FFPE samples have not been compatible with long-read sequencing. That’s changing. This year at AACR, we shared how the Ampli-Fi protocol is making it possible to use FFPE tissue with HiFi sequencing, opening up access to a massive collection of samples that were previously too degraded to use. From phasing somatic mutations to detecting structural variants, this shift could help researchers ask better questions (and get better answers) about how cancer behaves, adapts, and responds to treatment.
What is FFPE and why does it matter in cancer research?
As one of the most common approaches in cancer research, FFPE is a Formalin-Fixed Paraffin-Embedded preservation method and has been the clinical standard for decades. This method stabilizes tissue structure for long-term storage and histological analysis, but its chemical fixation and embedding process results in DNA damage and fragmentation, so that only very few long, intact molecules remain. FFPE samples have thereby posed a significant sample type barrier for long-read sequencing.
Why unlocking FFPE is a big deal
And yet, FFPE samples represent one of the most abundant and valuable resources in cancer research. They allow scientists to study real patient tumors often with rich clinical context and enable retrospective studies that span years or even decades. They’re also the foundation for many translational and clinical genomics programs, where the ability to profile preserved tumor tissue can influence treatment decisions or identify new biomarkers. For these reasons, FFPE tissue has remained a top priority for researchers despite the technical challenges.
Introducing the Ampli-Fi protocol for FFPE HiFi sequencing
Recognizing the enormous potential locked in FFPE samples, PacBio aimed to bring HiFi sequencing to this previously inaccessible sample type. The release of the Ampli-Fi protocol earlier this year now opens the door to HiFi sequencing for challenging sample types like FFPE. Ampli-Fi is a new workflow that enables HiFi sequencing from ultra-low DNA inputs, from as little as 1 ng of genomic DNA. With a streamlined one-day prep, Ampli-Fi represents a major leap forward in the accessibility of HiFi sequencing.
How Ampli-Fi enables FFPE sequencing without sacrificing quality
This ultra-low input enabled by Ampli-Fi, now unlocks fragmented DNA found in FFPE tissue for HiFi sequencing, without sacrificing the quality of the reads. Further results may vary depending on sample quality, with higher-DIN samples yielding longer read lengths, but initial sequencing of FFPE samples from the EFGR cell line and breast and lung tissue confirm this exciting possibility: FFPE samples sequenced on the Revio system with SPRQ chemistry and the Ampli-Fi protocol show higher Q scores than ever.
Libraries prepared from FFPE-extracted DNA yield high-quality HiFi reads with median read quality up to Q60 and mean read lengths in the 2–3 kb range. While not as long as standard HiFi reads, this read length is sufficient to deliver powerful insights into variant phasing and structural variation — two areas where HiFi sequencing offers unique advantages.
Full-gene phasing now possible in archived tumor samples
In EGFR-mutant FFPE samples, HiFi sequencing successfully phased the entire ~200 kb EGFR gene. All variants within this gene could be assigned to parental haplotypes, critical for identifying compound heterozygous variants, where pathogenic mutations on both alleles of a gene can disrupt its function. This precise phasing capability is especially important for cancer research, where determining whether two variants are in cis (on the same chromosome) or in trans (on opposite chromosomes) can be crucial for understanding their functional consequences for tumor progression and drug resistance.
When evaluating phasing across known cancer genes ≤50 kb in size, 68% could be phased with a single phase block using FFPE-derived libraries prepared with Ampli-Fi. For comparison, standard libraries from high-quality DNA achieved 84% — only a small drop given the fragmented nature of FFPE DNA. These findings highlight how even modest HiFi read lengths from degraded material can still deliver full-gene phasing in a majority of cancer-relevant targets.
Structural variant detection in FFPE tissue
Another HiFi advantage brought to FFPE? Structural variant detection. In a lung cancer FFPE sample, a 310 bp Alu insertion was detected in an intron of the PTPRD gene, a tumor suppressor mutated in multiple cancer types1. This insertion was not only identified but also phased within an 8 kb region, showing how even relatively short HiFi reads (2–3 kb) can provide critical structural context. These types of variants can be biologically significant in cancer, altering gene regulation or disrupting key domains—and detecting them from archived samples opens a new window into tumor evolution.
While the Ampli-Fi protocol is already breaking barriers for challenging samples, there’s even more room to grow. Early testing suggests that combining Ampli-Fi with Kinnex concatenation can significantly increase coverage, further enabling the ability to uncover important cancer variants in FFPE samples even at low frequency.
Key takeaways: FFPE with HiFi is now a reality
These early results prove what was once considered a fundamental barrier for FFPE samples is no longer a limitation. With the Ampli-Fi protocol, we’re seeing the power of these conclusions:
- HiFi sequencing of FFPE samples is now possible using the Ampli-Fi‑ protocol
- FFPE sequencing yields exceptional Q scores and despite some yield variation due to sample quality, sufficiently long reads
- Variant phasing across entire cancer genes is achievable, enabling cis/trans analysis and identification of compound heterozygosity
- Structural variants can be resolved from FFPE samples that short reads struggle with
A milestone for cancer genomics and translational research
Bringing HiFi sequencing to FFPE samples is a milestone that changes what’s possible for cancer genomics. With Ampli-Fi, PacBio is making it possible to generate high-quality, information-rich sequencing data from preserved cancer tissues. That means researchers can revisit biobanked samples, analyze tumors from clinical studies, and access previously untapped genomic information that may hold the key to new treatments or diagnostics.
To learn more about how HiFi sequencing can transform cancer research, download the brochure and stay tuned for an FFPE webinar coming this summer.