Single-Spore Genome Sequencing Goes High-Throughput With SAG-gel Platform for Filamentous Fungi

Fungal genomes have long been a story of the accessible few. The vast majority of known fungal diversity lives in environmental samples, in rare taxa, or in organisms that stubbornly refuse to grow in culture, and traditional genome sequencing has only ever captured the species that can be coaxed into producing grams of mycelial biomass. Now a team at Waseda University in Tokyo reports a workflow that sidesteps that bottleneck entirely: they have adapted a single-cell genomics platform to sequence the genomes of individual fungal spores, one spore at a time, at a scale that previous single-cell fungal approaches never reached. The study, published in BMC Genomics, demonstrates high-throughput recovery of partial genomes from single spores of filamentous ascomycete fungi using a technique known as SAG-gel, short for single-amplified genome in gel.

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