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

Marisol Sánchez-García,Verena Esther Kutschera,Barbara Ellis,Anna Rosling,Marcin Kierczak,Merce Montoliu-Nerin,Manfred Grabherr,Claudia Bergin,Hanna Johannesson

doi:10.1038/s41598-020-58025-3

Marisol Sánchez-García, Verena Esther Kutschera + Show 7 more

Open Access

https://doi.org/10.1038/s41598-020-58025-3

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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.

Highlights

The advent of novel sequencing techniques has unraveled a tremendous diversity on Earth
The different assembly workflows resulted in assemblies that vary in size, fragmentation and completeness (Table 1)
Of the core single copy genes identified by BUSCO, few were fragmented or duplicated in assembly 3n indicating that the set of 14,600 predicted genes is likely to be complete and a close representation of the genetic content in this strain (Table 1)

Summary

Introduction

The advent of novel sequencing techniques has unraveled a tremendous diversity on Earth. The development of metagenomic methods[6,7] and the advent of single cell sequencing[8,9,10] have revolutionized the study of life and function of cryptic organisms by upending the need for large and pure biological material, and allowing generation of genomic data from complex or limited environmental samples. This workflow allows, for the first time, the generation of reference genome assemblies from large scale, unbiased sorted, and sequenced AM fungal nuclei, circumventing tedious and often impossible culturing efforts This method opens infinite possibilities for studies of evolution and adaptation in these important plant symbionts and demonstrates that reference genomes can be generated from complex non-model organisms by isolating only a handful of their nuclei. The MDA method, has an advantage over PCR-based methods in that it produces longer fragments of DNA with a lower error rate and random coverage bias[36,37]

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Results

Conclusion