The logic behind targeting spores is elegant. In fungi, spores are discrete biological units of dispersal and persistence, essentially self-contained packages of a haploid or dikaryotic genome that can survive in soil, water, and air. Each spore is, in effect, a natural single cell. If researchers could crack open a single spore, amplify its DNA, and sequence it, they could in principle recover genome information from organisms that never need to be cultured at all. That capability matters because amplicon surveys and metagenomics, the workhorses of environmental fungal diversity studies, reveal which taxa are present but do not preserve the genome context of individual propagules, and recovering low-abundance fungal genomes from complex microbial communities remains notoriously difficult.

The SAG-gel platform, developed previously for bacterial single-cell genomics, works by encapsulating individual cells in hydrogel beads before lysis and amplification. The gel matrix physically confines the amplified DNA while allowing reagents to diffuse in and out, which prevents the cross-contamination and loss of genetic material that plague conventional tube-based single-cell workflows. Adapting this system to fungi posed a distinct challenge: fungal spores are encased in tough, chitin-rich cell walls that are far more resistant to chemical lysis than bacterial membranes. The researchers, Nevin McCone, Zikai Xiang, and Masahito Hosokawa, therefore systematically benchmarked lysis conditions, and found that a cocktail containing Yatalase, an enzyme blend that degrades chitin and other fungal cell wall polysaccharides, produced the highest proportion of amplification-positive spores among all the conditions they tested.

With lysis optimized, the team validated the workflow on cultured filamentous ascomycetes, choosing Aspergillus niger, the industrial workhorse that produces citric acid and a suite of enzymes, and Colletotrichum nagasakiense, a plant-pathogenic species, as their primary test organisms, with additional validation in Neurospora crassa, the classic genetic model fungus. Using fluorescence-activated cell sorting, they sorted 192 amplification-positive gel beads per species into individual reactions. In the direct workflow, in which the gel-encapsulated first-round amplification products, generated by multiple displacement amplification or MDA, were used straight for sequencing library preparation, 175 Aspergillus niger and 170 Colletotrichum nagasakiense single-amplified genomes passed quality control. That success rate represents a level of throughput that single-spore sequencing has simply not achieved before.

The quality of the individual assemblies, however, tells a more nuanced story. Each single-spore genome remained partial, with mean reference-genome breadths of 51.2 percent for Aspergillus niger and 38.0 percent for Colletotrichum nagasakiense. This incompleteness is a well-known consequence of MDA, the strand-displacement amplification chemistry at the heart of nearly all single-cell genomics. MDA copies DNA with high yield but unevenly, generating deep coverage over some regions of the genome while leaving others entirely unamplified, a phenomenon called amplification bias. For a single spore, whose DNA content is minuscule, that bias means a substantial fraction of genes will be missing from any individual assembly, no matter how deeply the amplified DNA is sequenced.

The researchers also implemented an indirect workflow, in which the amplified DNA from gel beads is pooled and re-amplified to generate microgram-scale quantities of DNA before library preparation. This approach delivered greater sequencing depth and produced an archive of amplified material that can be stored and resequenced later, a practical advantage for rare or precious samples. But in the present comparison, the indirect route did not yield broader genome recovery than the direct route, and after read counts were matched between the two workflows, the indirect assemblies showed higher coverage inequality, suggesting that the additional amplification rounds amplify the existing bias rather than correcting it.

The most striking result came from combining genomes. When the team co-assembled twelve direct-workflow single-amplified genomes derived from the same cultured strain, the composite assemblies reached approximately 97 percent BUSCO completeness for Aspergillus niger and 90 percent for Colletotrichum nagasakiense. BUSCO, the Benchmarking Universal Single-Copy Orthologs metric, measures how many conserved single-copy genes are present in an assembly, and values above 90 percent are generally considered near-complete for genome quality assessment. In other words, while no single spore yielded a complete genome, a modest panel of spores from the same population collectively covered nearly the entire genome. Indirect-workflow co-assemblies, by contrast, showed lower recovery and higher fragmentation in the primary species comparison, reinforcing the direct workflow as the stronger option for maximizing recovered sequence.

This co-assembly strategy points toward how the platform would actually be used in practice. For environmental or clinical samples, researchers would sort many individual spores, sequence each one, and then bin together the single-amplified genomes that share sequence similarity, effectively reconstructing strain-level genomes from organisms that were never cultured. Because each SAG originates from a single propagule, the approach preserves the linkage between genes within one genome, something metagenomic assembly struggles to guarantee in communities full of closely related fungal strains. That property is particularly valuable for resolving population structure among plant pathogens, for identifying biosynthetic gene clusters in rare environmental taxa, and for connecting functional genes to specific lineages in complex mycobiomes.

The authors are careful about the boundaries of what they have shown. All three test species belong to the Pezizomycotina, the largest subphylum of ascomycete fungi, and the researchers note that applicability beyond this group remains to be established. Basidiomycete spores, early-diverging fungal lineages, and spores with especially recalcitrant walls may require different lysis chemistries. Two technical fronts stand out as priorities for future work: improving spore-compatible lysis so that a larger fraction of sorted spores release their full DNA content, and improving the uniformity of the first-round amplification so that individual SAGs cover more of their genome. Progress on either front would directly increase the genome breadth recoverable from each single spore and reduce the number of spores needed for a near-complete assembly.

Even with those caveats, the study marks a meaningful step for fungal genomics. Fungal genomic resources remain heavily biased toward taxa that can be cultured, enriched, or isolated in sufficient biomass, and this work offers a complementary path that scales. The direct and indirect implementations of SAG-gel give laboratories two distinct practical options: the direct route for scalable sequencing of many spores, and the indirect route for generating archival amplified DNA from samples that may never be recovered again. As the workflow matures, the prospect of sequencing genomes directly from spores captured out of soil, air, or plant tissue moves from aspiration toward routine practice, opening a genomic window onto the fungal dark matter that culture-based methods have left in the shadows.

Subject of Research: High-throughput single-spore genome sequencing of filamentous ascomycete fungi using the SAG-gel single-cell genomics platform

Article Title: High-throughput single-spore genome sequencing of filamentous ascomycete fungi using the SAG-gel platform

Article References: McCone, N., Xiang, Z., & Hosokawa, M. (2026). High-throughput single-spore genome sequencing of filamentous ascomycete fungi using the SAG-gel platform. BMC Genomics. https://doi.org/10.1186/s12864-026-13433-z

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13433-z

Keywords: single-cell genomics, fungal spores, SAG-gel, multiple displacement amplification, genome assembly, Aspergillus niger, Colletotrichum nagasakiense, Neurospora crassa, filamentous ascomycetes, fluorescence-activated cell sorting, BUSCO, fungal genomics

The core idea is elegantly simple. A fluorescent tracer dye called Fast Blue is applied to the organ of interest. Neurons whose axons terminate in that organ take up the dye at their nerve endings and transport it backward along their axons to their cell bodies, a process known as retrograde axonal transport. A few days later, the relevant ganglia are dissected, dissociated into single cells, and passed through a fluorescence-activated cell sorter. Only the fluorescently labeled neurons, those with a proven physical connection to the target tissue, are retained. Each captured cell is then subjected to single-cell RNA sequencing, generating a full transcriptome for every traced neuron.

The crucial advance over existing approaches lies in selectivity. Sequencing a whole ganglion without labeling captures everything: sensory neurons serving skin and muscle, autonomic neurons innervating unrelated organs, satellite glial cells and other stromal populations. That background can completely swamp the small subset of neurons connected to the tissue a researcher actually cares about. Bulk sequencing is even blunter, averaging signals across thousands of cells and erasing neuronal subtype identity altogether. Trace-n-Seq eliminates this noise by design, allowing researchers to characterize organ-specific neurons at single-cell resolution and to detect even rare tissue-innervating subsets that would otherwise be invisible.

The complete workflow, published in Nature Protocols by Vera Thiel, Manuel Mastel, Simon Renders and colleagues in the laboratories of Martin Sprick and Andreas Trumpp, can be completed in about two weeks. It encompasses tracer application, ganglion dissection, enzymatic tissue digestion, careful neuronal validation, fluorescence-based sorting, SMART-seq2 library preparation and a dedicated bioinformatic pipeline. The published protocol walks readers through each stage with schematic illustrations, dissection guidance, quality-control checkpoints and computational scripts. The authors note that the method suits researchers experienced in molecular biology, neuronal tissue handling, sequencing techniques and bioinformatics, and they have deposited their complete Seurat-based analysis workflow, quality-control scripts and annotation code openly on GitHub. The underlying sequencing data are accessible through the ArrayExpress repository under accession E-MTAB-12940.

Why does this matter biologically? Neuronal innervation is now recognized as a master regulator of tissue function well beyond classical sensation and motor control. Nerves shape organ function during homeostasis and regeneration, and they are increasingly implicated in pathological settings including inflammation, autoimmune disease, fibrosis and cancer. The emerging field of cancer neuroscience, highlighted in the protocol’s own key reference by Monje and Winkler, argues that tumors are not just passive targets of nerve growth but active participants in a dialogue with the nervous system. Tumor-associated nerves can be reprogrammed by the tumor microenvironment, and in some contexts these remodelled neurons appear to support malignancy itself.

Trace-n-Seq was developed and validated in exactly such a context. The protocol builds on the team’s earlier research published in Nature in 2025, in which they characterized single neurons reprogrammed by pancreatic cancer. Pancreatic ductal adenocarcinoma is one of the most densely innervated human tumors, and the earlier study showed that individual neurons connected to the tumor acquire distinct transcriptional states compared with neurons innervating healthy tissue. By tracing neurons from the tumor back to their ganglia and sequencing them one by one, the researchers could separate genuine disease-associated neuronal states from the bulk signature of the ganglion. The new protocol packages that experimental logic into a step-by-step recipe that any qualified laboratory can follow.

The versatility of the method is a major selling point. Because it depends only on functional connectivity, Trace-n-Seq is in principle applicable to any healthy, inflamed or diseased tissue innervated by the peripheral nervous system. Researchers can compare traced neurons from a healthy pancreas with those from a pancreatitis model or a tumor-bearing organ, directly quantifying how disease remodels neuronal subtype composition and gene expression. They can capture rare tissue-specific neuronal subsets, distinguish disease-associated states from background ganglia signatures, and follow neuronal plasticity across physiological and pathological conditions at single-cell resolution. This functional, connectivity-based profiling contrasts with purely anatomical or genetic labeling strategies and provides an orthogonal view of peripheral neuron diversity.

The technical details matter for anyone hoping to reproduce the approach. Fast Blue is a retrograde tracer well established in neuroanatomy, valued for its intense fluorescence and compatibility with downstream flow cytometry. After allowing sufficient time for axonal transport, ganglia are harvested and must be gently digested to yield viable, intact neurons, a step the authors emphasize with dedicated validation procedures to confirm that sorted cells are indeed neurons rather than debris or contaminating glia. Fluorescence-activated cell sorting then enriches the labeled population before library construction. The bioinformatic workflow includes quality control, filtering and annotation steps tailored to traced neurons, including reference-based neuronal annotation, so that researchers can confidently classify traced cells into known sensory and autonomic subtypes and discover novel disease-associated clusters.

The publication arrives at a moment of rapid growth for the neuro-immune and neuro-oncology fields. Other studies have revealed, for example, that lymph nodes are innervated by a unique population of sensory neurons with immunomodulatory potential, and barcoded viral tracing approaches have begun to map single-cell interactions during central nervous system inflammation. Large-scale atlases from single-cell sequencing efforts have catalogued the molecular architecture of the mouse nervous system, classifying sensory and visceral motor neuron types in unprecedented detail. What these atlases lack, however, is information about where each neuron actually projects. Trace-n-Seq adds that anatomical dimension, linking molecular identity to functional tissue connectivity in a single experiment.

For clinical translation, the implications are considerable. If neurons innervating tumors can be identified and molecularly profiled, they become potential therapeutic targets or biomarkers. Clinically actionable strategies for studying neural influences in cancer have been a subject of active discussion, and tools like Trace-n-Seq provide the granular data needed to move from observation to intervention. Understanding which neuronal subprograms a tumor co-opts, and how inflammatory or fibrotic diseases rewire peripheral innervation, could eventually inform drugs that interrupt harmful neuro-tissue signaling while sparing essential nerve function. The open release of the protocol, the analysis code and the source datasets lowers the barrier for laboratories worldwide to adopt the technique, and the authors declare no competing interests. As neuroscience and organ biology continue to converge, methods that reveal which neurons talk to which tissues, and what they say at the transcriptomic level, are likely to become standard equipment in the effort to understand how the nervous system shapes health and disease.

Subject of Research: A single-cell sequencing protocol combining retrograde fluorescent tracing and cell sorting to molecularly profile peripheral neurons innervating specific tissues.

Article Title: Trace-n-Seq combines retrograde fluorescent tracing with sorting and single-cell sequencing of innervating peripheral neurons

Article References: Thiel, V., Mastel, M., Renders, S., Panten, J., Bauer, K., Jackstadt, R., Sprick, M. R., & Trumpp, A. (2026). Trace-n-Seq combines retrograde fluorescent tracing with sorting and single-cell sequencing of innervating peripheral neurons. Nature Protocols. https://doi.org/10.1038/s41596-026-01416-z

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01416-z

Keywords: Trace-n-Seq, retrograde tracing, single-cell RNA sequencing, peripheral neurons, fluorescence-activated cell sorting, ganglia, cancer neuroscience, neuronal innervation, Fast Blue, pancreatic cancer, Nature Protocols, neuron-tissue interactions