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

Abstract Background Fungal genomic resources remain biased toward taxa that can be cultured, enriched, or isolated in sufficient biomass. Amplicon and metagenomic surveys reveal extensive diversity but do not preserve the genome context of individual propagules, and recovery of low-abundance fungal genomes from complex samples remains difficult. Fungal single-cell genomics provides a complementary approach, but current workflows remain limited in throughput, taxonomic scope, and sample requirements. Unicellular spores provide a practical target because they are discrete biological units of dispersal and persistence. Results We adapted and benchmarked a single-amplified genome in gel (SAG-gel) workflow for high-throughput genome sequencing of individual fungal spores. The workflow was tested with cultured filamentous ascomycetes: Aspergillus niger and Colletotrichum nagasakiense as primary species, with additional validation in Neurospora crassa . Yatalase-containing lysis conditions produced the highest amplification-positive recovery among the conditions tested. In the direct workflow, gel-encapsulated first-round MDA products were used directly for library preparation; 175 A. niger and 170 C. nagasakiense SAGs passed quality control from 192 sorted amplification-positive gel beads per species. Individual direct-workflow assemblies remained partial, with mean reference-genome breadths of 51.2% in A. niger and 38.0% in C. nagasakiense . The implemented indirect workflow generated microgram-scale amplified DNA and greater sequencing depth, but did not yield broader genome recovery in the present comparison and showed higher coverage inequality after read-count matching. Co-assembly of 12 direct-workflow SAGs obtained from the same cultured strain increased BUSCO completeness to approximately 97% in A. niger and 90% in C. nagasakiense . Indirect-workflow co-assemblies showed lower recovery and higher fragmentation in the primary species comparison. Conclusions SAG-gel supports high-throughput recovery of partial single-spore SAGs from the cultured filamentous ascomycetes tested here. The direct and indirect implementations provide distinct practical options for scalable sequencing and recovery of archival amplified DNA, respectively. Improving spore-compatible lysis and first-round amplification uniformity remains central to increasing individual-SAG genome recovery, and applicability beyond the tested Pezizomycotina remains to be established.