Formalin-fixed, paraffin-embedded (FFPE) tissues represent the predominant sample conservation method in clinical practice, yet degraded and crosslinked RNA has long limited whole-transcriptome analysis and spatial context.
August 28, in Cell, researchers at BGI-Research and collaborating clinical and research centers published Stereo-seq V2, a spatial transcriptomics method that achieves single-cell–level resolution on FFPE sections and, critically, enables simultaneous in situ profiling of host and microbial RNAs. By combining deparaffinization/decrosslinking with random-primed capture and uniform gene-body coverage, Stereo-seq V2 opens FFPE "black boxes" for infectious disease biology, tumor ecosystems, and clinical research applications.
[Online paper screenshot]
The study “Stereo-seq V2: Spatial mapping of total RNA on FFPE sections with high resolution” was published in Cell.
Stereo-seq V2 workflow enables simultaneous host and microbial RNA mapping on FFPE tissues with single-cell resolution.
Built on the large field of view and subcellular spot spacing of Stereo-seq, the V2 chemistry replaces poly(T) capture with random primers, increasing sensitivity to low-abundance and non-poly(A) RNAs while maintaining signal-to-noise. This advance enables sharper boundaries and cellular resolution, with V2 reducing lateral diffusion at tissue borders and achieving true single-cell segmentation on FFPE sections, identifying 34 cell types in mouse brain. Across adjacent mouse brain sections, V2 and V1 showed high concordance in gene expression and reduced apparent lateral diffusion at anatomical borders.
Compared with existing probe-based methods, V2 provides whole-transcriptome coverage, significantly expanding beyond traditional probe limitations, while recovering important markers and regulatory genes otherwise missed or inconsistently captured by probe panels. Uniform coverage across exons improves detection of transcription factors, adhesion genes, and alternative splicing events, categories that often prove elusive with 3′-biased strategies.
Stereo-seq V2 demonstrates improved spatial resolution with reduced lateral diffusion at tissue boundaries and achieves single-cell resolution on FFPE sections, identifying 34 distinct cell types in mouse brain.
Demonstrating clinical FFPE robustness, V2 maintains high gene capture even on severely degraded samples and aligns molecular signatures with histological features like necrosis. V2 performs robustly on clinical FFPE samples with poor RNA integrity. In a cohort of triple-negative breast cancer (TNBC) blocks preserved from under 1 to ~9 years, V2 maintained consistent gene capture. The method showed reliable performance across different tissue conditions.
Spatial expression recapitulated clinical immunohistochemical phenotypes (e.g., low ESR1/PGR/ERBB2), delineated malignant, immune, and necrotic regions, and supported tumor subtype classification through genetic analysis. Whole gene-body coverage enabled spatial alternative splicing analysis, identifying 1,492 events including specific splicing patterns in tumor-associated genes like ZNF226, GIPC1, and BEX4 that varied between tumor subtypes. These findings add molecular granularity to tumor biology beyond expression alone.
V2 maintains robust gene capture across degraded clinical TNBC samples and accurately maps tumor, immune, and necrotic regions.
A core advantage is unbiased, concurrent mapping of host and microbial RNAs directly on FFPE tissue, enabling host–microbe battlefield mapping where V2 simultaneously maps spatial B-cell receptor (BCR) clones in human Tuberculosis (TB) lungs and tracks Mtb infection dynamics with bacterial RNA peaking at 4 weeks in mouse models.
In a Mycobacterium tuberculosis (Mtb) mouse model profiled at 1 day, 4 weeks, and 8 weeks post infection, V2 captured the rise-and-fall kinetics of bacterial transcripts consistent with histology and CFU counts and identified host gene modules spatially correlated with Mtb burden, shifting from inflammatory and cell death pathways to adaptive immunity over time.
tereo-seq V2 further resolves the in situ BCR repertoire. Compared with poly(A)-based methods, V2 provides markedly improved coverage of V regions, enabling assembly of clonotypes and analysis of somatic hypermutation. The number of assembled BCR clones increased from 185 at 4 weeks to 1,736 at 8 weeks, with greater clonal diversity and mutation frequency proximal to infected regions, consistent with affinity maturation.
In human TB FFPE lung samples, V2 similarly mapped host and pathogen RNAs, delineated necrotic boundaries from RNA density, and revealed recurrent BCR clones across patients; related clones were enriched in blood from active TB cases, pointing to potential diagnostic and therapeutic targets.
Spatial BCR repertoire analysis reveals clonal expansion and affinity maturation in TB-infected tissues, with related clones detected in patient blood.
Together, these results position Stereo-seq V2 as a broadly applicable platform for single-cell–level, whole-transcriptome mapping on FFPE sections, extending spatial transcriptomics to the most abundant and clinically relevant specimens. By unifying host gene programs, immune repertoires, and pathogen localization within the same section, V2 provides a panoramic readout of tissue ecosystems in cancer, infection, and beyond, and may accelerate retrospective biomarker discovery, mechanism-based stratification, and antibody or vaccine development using existing archives.
All human and animal studies were conducted with appropriate institutional ethics approvals and followed established guidelines for research involving clinical specimens and laboratory animals. Data and code availability: Raw data are deposited in the Genome Sequence Archive; analysis code is available at the project repository: GitHub (Stereo-seq V2 code) at https://github.com/YoungLi88/Stereo-seq-V2. The study can be accessed here: xxxxxxx