A closed Candidatus Odinarchaeum genome exposes Asgard archaeal viruses

Asgard archaea have recently been identified as the closest archaeal relatives of eukaryotes. Their ecology remains enigmatic, and their virome, completely unknown. Here, we describe the closed genome of Ca. Odinarchaeum yellowstonii LCB_4, and, from this, obtain novel CRISPR arrays with spacer targets to several viral contigs. We find related viruses in sequence data from thermophilic environments and in the genomes of diverse prokaryotes, including other Asgard archaea. These novel viruses open research avenues into the ecology and evolution of Asgard archaea.


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Asgard archaea are a diverse group of microorganisms that comprise the closest relatives of 28 eukaryotes 1-7 . Their genomes were first explored over six years ago 8 , and much of their 29 physiology and cell biology remains to be studied. While over two hundred draft genomes are 30 available for this group, the majority is represented by highly fragmented and incomplete 31 metagenome assembled genomes (MAGs), which has precluded obtaining insights into their

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To obtain a complete Asgard archaeal genome, we reassembled the Odinarchaeota LCB_4 36 genome, currently a 96% complete assembly of 1.46 Mbp distributed in 9 contigs 1 . A promising 37 reassembly yielded a 1.41 Mbp contig, a 13 kbp contig containing CRISPR-associated (cas) 38 genes, and multiple short contigs harboring mobile element or repeat signatures (see Methods

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for details). After contig boundary inspection, we postulated that the first two contigs 40 represented the entire Odinarchaeota LCB_4 chromosome as these were flanked by similar 41 CRISPR arrays that extended for several kilobasepairs (Suppl. Fig S1). We successfully 42 amplified these gaps using long-range PCR, sequenced the resulting amplicons with 43 Nanopore sequencing, and performed a hybrid assembly, finally generating a single 1.418 44 Mbp circular contig. Given the high quality of this genome, we suggest recognizing this strain 45 as Candidatus Odinarchaeum yellowstonii LCB_4 (hereafter 'LCB_4'), in reference to 46 Yellowstone National Park, location of the hot spring where it was sampled.

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The LCB_4 genome contains a unique disposition of CRISPR-Cas genes (Fig. 1), including 48 neighboring Type I-A and Type III-D cas gene clusters, separated by a 6.1 kb-long Type I-A 49 CRISPR array, and further followed by another 2.7 kb-long Type I-A CRISPR array, with a 50 total of 144 CRISPR spacers across both arrays. Nine of these spacers targeted, with 100% 51 identity and query coverage, four putative mobile element contigs obtained in the same 52 assembly that were not part of the closed genome ( Fig. 1). Two of these contigs contained 53 genes encoding common mobile element proteins, such as restriction endonucleases and 54 integrases, but did not contain any viral signature genes (Suppl.   Table S1). This specific protein 58 was previously found in a study of the DJR-MCP family, and was tentatively classified as 59 belonging to an "Odin group" since it was found in the same metagenome as Ca.

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Odinarchaeum LCB_4 9 . The present complete recovery of LCB_4 CRISPR arrays allowed us 61 to confirm that this circular contig indeed represents a virus associated with Ca.

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Odinarchaeum, for which we suggest the name Huginnvirus, in reference to Odin's raven,

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Long-range PCR and Nanopore sequencing

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Four regions were selected for long-range Polymerase Chain Reaction (lrPCR): two contig 187 gaps, corresponding to CRISPR arrays, and two control regions spanning ca. 5 kb of the rRNA operon and ca. 10 kb of a ribosomal protein gene cluster (Suppl.

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Amplification of control and gap regions was then performed following the parameters shown 194 in Suppl. Table S2. Products were separated on a 0.8% agarose gel in 1XTBE stained with

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Hybrid assembly

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Reads were filtered using NanoFilt v.2.6.0 with options "-q 10 -l 1000". We used these filtered 208 Nanopore reads and the mapped Illumina reads to perform a hybrid assembly with Unicycler