Building de novo reference genome assemblies of complex eukaryotic microorganisms from single nuclei

Abstract

The advent of novel sequencing techniques has unraveled a tremendous diversity on Earth. Genomic data allow us to understand ecology and function of organisms that we would not otherwise know existed. However, major methodological challenges remain, in particular for multicellular organisms with large genomes. Arbuscular mycorrhizal (AM) fungi are important plant symbionts with cryptic and complex multicellular life cycles, thus representing a suitable model system for method development. Here, we report a novel method for large scale, unbiased nuclear sorting, sequencing, and de novo assembling of AM fungal genomes. After comparative analyses of three assembly workflows we discuss how sequence data from single nuclei can best be used for different downstream analyses such as phylogenomics and comparative genomics of single nuclei. Based on analysis of completeness, we conclude that comprehensive de novo genome assemblies can be produced from six to seven nuclei. The method is highly applicable for a broad range of taxa, and will greatly improve our ability to study multicellular eukaryotes with complex life cycles.

Identifying the causes and consequences of assembly gaps using a multiplatform genome assembly of a bird‐of‐paradise

“…Additionally, the large number of assembly tools available and hundreds of parameters to tweak makes it inevitable to produce numerous different assembly versions. For example, we generated 15 different assemblies only for the parameter optimization of the linked-read scaffolding ( Figure 1d) and there are studies generating even 400 assemblies in total (Montoliu-Nerin et al, 2020). In such a situation, it might seem difficult to decide how to choose the "best" assembly among dozens.…”

Current State and Future Directions of Genetics and Genomics of Endophytic Fungi for Bioprospecting Efforts

“…The major difficulty in using this technology on endophytic fungi is probably the preparation of single cells. The two studies applying single-cell genomics to fungi used spores that can easily be harvested from cultivable fungi (Ahrendt et al, 2018 ; Montoliu-Nerin et al, 2020 ). However, for uncultivable organisms this is not possible and other strategies need to be developed.…”

“…However, for uncultivable organisms this is not possible and other strategies need to be developed. Looking at the recent advances in plant single-cell genomics (Luo C. et al, 2020 ), the most promising strategies, will most likely be the preparation of protoplasts by cell wall digestion (host plant and fungal cells need to be digested), and/or the isolation of nuclei (Montoliu-Nerin et al, 2020 ). Once a suspension of cells or nuclei is prepared, they need to be separated by manual sorting or by Fluorescence-activated cell sorting (FACS).…”

In-depth phylogenomic analysis of arbuscular mycorrhizal fungi based on a comprehensive set of de novo genome assemblies

“…Single nuclei were sorted, followed by whole genome amplification (WGA) and sequencing. Finally, the data from several nuclei were combined to build a de novo genome assembly of Claroideoglomus claroideum (Montoliu-Nerin et al, 2020). With this novel workflow AM fungal genome assemblies can be obtained from as little as one single spore, independently of the species ability to grow in axenic cultures.…”