Presentation Information
[P03-362]Development of a whole-genome sequencing method for single fungal spores using droplet microfluidics
○Nevin McCone1, Zikai Xiang2, Kazuki Takahashi3, Masahito Hosokawa1,2,3,4 (1. Grad. Sch. Adv. Sci. Eng., Waseda Univ. (Japan), 2. Research Institute for Science and Engineering, Waseda Univ. (Japan), 3. Institute for Nanoscience and Nanotechnology (Nano & Life Innovation), Waseda Univ. (Japan), 4. Institute for Advanced Biomedical Sciences (Institute for Life Dynamics Research), Waseda Univ. (Japan))
Keywords:
Fungal spores,Single-cell sequencing,Droplet microfluidics,Microbial genomics
[Purpose]: Accurate fungal genome characterization is essential for industrial applications. However, it remains impossible to culture most fungal species in the laboratory, limiting access to the full genomic content of uncultured fungi. Bulk metagenomics presents a culture-free alternative by enabling sequencing of environmental samples but often struggles to capture low-abundance species and strain-level variations. Single-cell genomics methods attempt to overcome these limitations by isolating individual cells prior to sequencing. However, the minute amount of DNA available in a single cell and the resource investment required for processing individual cells present additional barriers. Therefore, this study aimed to develop a novel high-throughput single fungal spore whole-genome sequencing method using droplet microfluidics.
[Method]: Single spores from four cultured fungal strains (C. nagasakiense, A. niger, N. crassa, and V. dahliae) were encapsulated within agarose gel capsules using a droplet-based platform, known as “SAG-gel” (Hosokawa et al. Biophys. Rev. 2024) and exposed to a variety of enzymes with specific activity against key fungal cell wall components. Lysis conditions were evaluated by comparing the proportion of amplification-positive capsules after whole-genome amplification (WGA), as determined by SYBR Green staining. Multiple displacement amplification (MDA) was used as the WGA method. After WGA, capsules were individually sorted for sequencing library preparation. Sequencing reads were assembled in silico to generate single-amplified genomes (SAGs), which were then assessed for genome completeness and quality.
[Results]: The combination of multiple lytic enzymes, including yatalase, lyticase, and driselase, yielded the highest amplification-positive rate after WGA. Initial shallow sequencing of C. nagasakiense and A. niger yielded highly fragmented and incomplete SAGs. Although deep sequencing improved genome completeness, the resulting SAGs remained of low quality (<41% completeness). Reference mapping suggested that low-quality SAGs were a result of high levels of amplification bias. These results were consistent when sequencing was extended to N. crassa and V. dahliae. Co-assembly of multiple SAGs from the same species substantially improved genome completeness.
[Conclusion]: This study demonstrates that whole-genome sequencing from single fungal spores of diverse species is technically feasible and reproducible using droplet microfluidics. However, amplification bias remains a key limitation of MDA-based approaches, underscoring the need for strategies to mitigate this bias. Co-assembly of multiple SAGs is a promising approach for recovering high-quality genomes when multiple spores of the same species can be recovered from the same sample. The droplet-based platform presented opens a path toward culture-independent genomic exploration of the vast uncultured fungal diversity in natural environments.
[Method]: Single spores from four cultured fungal strains (C. nagasakiense, A. niger, N. crassa, and V. dahliae) were encapsulated within agarose gel capsules using a droplet-based platform, known as “SAG-gel” (Hosokawa et al. Biophys. Rev. 2024) and exposed to a variety of enzymes with specific activity against key fungal cell wall components. Lysis conditions were evaluated by comparing the proportion of amplification-positive capsules after whole-genome amplification (WGA), as determined by SYBR Green staining. Multiple displacement amplification (MDA) was used as the WGA method. After WGA, capsules were individually sorted for sequencing library preparation. Sequencing reads were assembled in silico to generate single-amplified genomes (SAGs), which were then assessed for genome completeness and quality.
[Results]: The combination of multiple lytic enzymes, including yatalase, lyticase, and driselase, yielded the highest amplification-positive rate after WGA. Initial shallow sequencing of C. nagasakiense and A. niger yielded highly fragmented and incomplete SAGs. Although deep sequencing improved genome completeness, the resulting SAGs remained of low quality (<41% completeness). Reference mapping suggested that low-quality SAGs were a result of high levels of amplification bias. These results were consistent when sequencing was extended to N. crassa and V. dahliae. Co-assembly of multiple SAGs from the same species substantially improved genome completeness.
[Conclusion]: This study demonstrates that whole-genome sequencing from single fungal spores of diverse species is technically feasible and reproducible using droplet microfluidics. However, amplification bias remains a key limitation of MDA-based approaches, underscoring the need for strategies to mitigate this bias. Co-assembly of multiple SAGs is a promising approach for recovering high-quality genomes when multiple spores of the same species can be recovered from the same sample. The droplet-based platform presented opens a path toward culture-independent genomic exploration of the vast uncultured fungal diversity in natural environments.
Comment
To browse or post comments, you must log in.Log in