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

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.

[1]  Michael Roberts,et al.  The MaSuRCA genome assembler , 2013, Bioinform..

[2]  Christina A. Cuomo,et al.  Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement , 2014, PloS one.

[3]  L. Farinelli,et al.  Ultra-low input transcriptomics reveal the spore functional content and phylogenetic affiliations of poorly studied arbuscular mycorrhizal fungi , 2017, DNA research : an international journal for rapid publication of reports on genes and genomes.

[4]  Cathy H. Wu,et al.  UniProt: the Universal Protein knowledgebase , 2004, Nucleic Acids Res..

[5]  S. Kingsmore,et al.  Comprehensive human genome amplification using multiple displacement amplification , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Martin Parniske,et al.  Arbuscular mycorrhiza: the mother of plant root endosymbioses , 2008, Nature Reviews Microbiology.

[7]  Lili He,et al.  Diversity and bioactive potential of culturable fungal endophytes of Dysosma versipellis; a rare medicinal plant endemic to China , 2018, Scientific Reports.

[8]  Christina A. Cuomo,et al.  Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth , 2012, Genome research.

[9]  Anders F. Andersson,et al.  Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea , 2011, The ISME Journal.

[10]  Jonathan E. Allen,et al.  Genome sequence of the human malaria parasite Plasmodium falciparum , 2002, Nature.

[11]  W. Koh,et al.  Single-cell genome sequencing: current state of the science , 2016, Nature Reviews Genetics.

[12]  T. James,et al.  Archaeorhizomycetes: Unearthing an Ancient Class of Ubiquitous Soil Fungi , 2011, Science.

[13]  S. Shigenobu,et al.  The genome of Rhizophagus clarus HR1 reveals a common genetic basis for auxotrophy among arbuscular mycorrhizal fungi , 2018, BMC Genomics.

[14]  J. Marleau,et al.  Spore development and nuclear inheritance in arbuscular mycorrhizal fungi , 2011, BMC Evolutionary Biology.

[15]  M. Rantalainen.  Application of single-cell sequencing in human cancer , 2017, Briefings in functional genomics.

[16]  T. James,et al.  No jacket required – new fungal lineage defies dress code , 2012, BioEssays : news and reviews in molecular, cellular and developmental biology.

[17]  Ying Li,et al.  Single Nucleus Genome Sequencing Reveals High Similarity among Nuclei of an Endomycorrhizal Fungus , 2014, PLoS genetics.

[18]  D. Edwards,et al.  Single-Cell Genomic Analysis in Plants , 2018, Genes.

[19]  R. Stepanauskas,et al.  Single-Cell Genomics Reveals Organismal Interactions in Uncultivated Marine Protists , 2011, Science.

[20]  Thijs J. G. Ettema,et al.  Asgard archaea illuminate the origin of eukaryotic cellular complexity , 2017, Nature.

[21]  P. Bonfante,et al.  Mechanisms underlying beneficial plant-fungus interactions in mycorrhizal symbiosis. , 2010, Nature communications.

[22]  Marcin Kierczak,et al.  From single nuclei to whole genome assemblies , 2019 .

[23]  Brian C. Thomas,et al.  Genome-reconstruction for eukaryotes from complex natural microbial communities , 2017, bioRxiv.

[24]  Brian C. Thomas,et al.  A new view of the tree of life , 2016, Nature Microbiology.

[25]  P. Franks,et al.  Mutualistic mycorrhiza-like symbiosis in the most ancient group of land plants. , 2010, Nature communications.

[26]  Alexey A. Gurevich,et al.  QUAST: quality assessment tool for genome assemblies , 2013, Bioinform..

[27]  Z. Fei,et al.  Genome and evolution of the arbuscular mycorrhizal fungus Diversispora epigaea (formerly Glomus versiforme) and its bacterial endosymbionts. , 2018, The New phytologist.

[28]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[29]  J. Lennon,et al.  Scaling laws predict global microbial diversity , 2016, Proceedings of the National Academy of Sciences.

[30]  T. Pawlowska,et al.  Multinucleate Spores Contribute to Evolutionary Longevity of Asexual Glomeromycota , 2010, The American Naturalist.

[31]  Jared T. Simpson,et al.  Exploring genome characteristics and sequence quality without a reference , 2013, Bioinform..

[32]  P. Lammers,et al.  Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis , 2013, Proceedings of the National Academy of Sciences.

[33]  M. Borodovsky,et al.  Gene prediction in novel fungal genomes using an ab initio algorithm with unsupervised training. , 2008, Genome research.

[34]  Lionel Guy,et al.  Exploring microbial dark matter to resolve the deep archaeal ancestry of eukaryotes , 2015, Philosophical Transactions of the Royal Society B: Biological Sciences.

[35]  G. Thor,et al.  Two Basidiomycete Fungi in the Cortex of Wolf Lichens , 2019, Current Biology.

[36]  B. Henrissat,et al.  High intraspecific genome diversity in the model arbuscular mycorrhizal symbiont Rhizophagus irregularis. , 2018, The New phytologist.

[37]  J. Banfield,et al.  Community structure and metabolism through reconstruction of microbial genomes from the environment , 2004, Nature.

[38]  Natalia N. Ivanova,et al.  Insights into the phylogeny and coding potential of microbial dark matter , 2013, Nature.

[39]  Brian C. Thomas,et al.  Critical biogeochemical functions in the subsurface are associated with bacteria from new phyla and little studied lineages. , 2016, Environmental microbiology.

[40]  J. McCutcheon,et al.  Basidiomycete yeasts in the cortex of ascomycete macrolichens , 2016, Science.

[41]  S. Kaul,et al.  “Omics” Tools for Better Understanding the Plant–Endophyte Interactions , 2016, Front. Plant Sci..

[42]  Evgeny M. Zdobnov,et al.  BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs , 2015, Bioinform..

[43]  B. Henrissat,et al.  Comparative genomics of Rhizophagus irregularis, R. cerebriforme, R. diaphanus and Gigaspora rosea highlights specific genetic features in Glomeromycotina. , 2019, The New phytologist.

[44]  Ning Ma,et al.  BLAST+: architecture and applications , 2009, BMC Bioinformatics.

[45]  Derrick E. Wood,et al.  Kraken: ultrafast metagenomic sequence classification using exact alignments , 2014, Genome Biology.

[46]  K. Sermon,et al.  Whole-genome multiple displacement amplification from single cells , 2006, Nature Protocols.

[47]  Todd H. Oakley,et al.  The Amphimedon queenslandica genome and the evolution of animal complexity , 2010, Nature.

[48]  A. Salamov,et al.  Leveraging single-cell genomics to expand the fungal tree of life , 2018, Nature Microbiology.

[49]  H. Friberg,et al.  New primers to amplify the fungal ITS2 region--evaluation by 454-sequencing of artificial and natural communities. , 2012, FEMS microbiology ecology.

[50]  Sofia M. C. Robb,et al.  MAKER: an easy-to-use annotation pipeline designed for emerging model organism genomes. , 2007, Genome research.

[51]  Miriah D. Meyer,et al.  Genome-wide synteny through highly sensitive sequence alignment: Satsuma , 2010, Bioinform..

[52]  Devin F R Doud,et al.  The trajectory of microbial single-cell sequencing , 2017, Nature Methods.

[53]  R. Lasken,et al.  Genomic DNA Amplification from a Single Bacterium , 2005, Applied and Environmental Microbiology.

[54]  S. Koren,et al.  Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation , 2016, bioRxiv.

[55]  Sergey I. Nikolenko,et al.  SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing , 2012, J. Comput. Biol..

[56]  Tanja Woyke,et al.  Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics , 2014, Nature Protocols.