Tobias Viehboeck1, Harald Gruber-Vodicka2, Patrick Hyden3, Thomas Rattei3, Silvia Bulgheresi1

Genomics of marine nematode symbionts Tobias Viehboeck1, Harald Gruber-Vodicka2, Patrick Hyden3, Thomas Rattei3, Silvia Bulgheresi1

- Laxus oneistus and Robbea hypermnestrae, two free-living nematodes (subfamily Stilbonematinae) live in a binary symbiotic association with chemoautotrophic, S-oxidizing Gammaproteobacteria, Candidatus Thiosymbion oneisti and Ca. T. hypermnestrae, respectively rchaea Biology and Ecogenomics - Each symbiont 9sattached to the nematode cuticle by one pole as to ensheath its host VIENNA

Laxus oneistus 150 µm Robbea hypermnestrae 150 µm

Ca. Thiosymbion oneisti 2 µm Ca. Thiosymbion hypermnestrae 1 µm

Illumina - Nanopore Hybrid assembly Single-cell sequencing - A single-worm metagenome was sequenced on Illumina HiSeq 2x100 bp, - FACS & WGA-XTM at the Bigelow SCGC assembled (SPAdes 3.11), binned with gbtools - 10 single-ampliﬁed genomes for sequencing on Illumina NextSeq 550 - A symbiont isolate was sequenced on a MinION (Oxford Nanopore 2x150 bp (12M reads/cell) Technologies, ONT) - thereof 3 for ONT sequencing

A A A A A A A A A A A B A A A A A A A A A A A B A A A A A A A A A A A B A A A A A A A A A A A B - The Illumina assembly was scaffolded with ultra long reads 03:23 04:02 08:55 02:03 05:30 03:22 03:41 02:00 06:56 02:08 02:48 02:04 02:05 02:04 03:30 02:04 02:29 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 09:24 03:57 02:25 03:19 02:22 02:24 02:14 02:06 02:33 02:45 03:24 02:47 02:03 01:57 02:30 02:35 01:44 10:18 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 10:28 09:04 06:18 05:11 09:21 07:26 02:11 02:23 02:44 02:43 04:00 02:51 03:08 02:37 02:09 02:05 02:13 02:12 07:30 02:51 02:17 02:25 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 09:30 11:02 09:14 02:31 06:34 02:32 03:38 04:15 02:03 11:05 01:51 02:02 02:06 02:49 08:19 3.6 B bases (Q34) 4 B bases (Q7) B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 08:22 03:56 10:13 09:59 10:52 06:16 05:30 02:40 03:45 02:57 05:48 02:28 01:49 02:23 05:53 03:34 02:12 07:31 02:07 02:54 02:08 01:49 02:37 02:25 02:06 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 09:03 08:22 06:56 02:08 02:27 02:11 03:14 02:05 08:24 01:53 02:07 02:07 02:05 02:52 02:06 09:04 36 M reads 48 K reads B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 01:52 04:07 06:53 10:29 04:34 06:55 08:45 04:50 04:56 01:39 02:56 01:55 01:56 04:12 02:01 02:12 01:57 04:59 01:39 05:55 03:07 04:11 01:45 01:47 02:35 01:59 C B B B B B B B B B B C B B B B B B B B B B B C C B B B B B B B B B B C B B B B B B B B B B B C 100 bp paired-end reads N50 read length: 1091 bp (max. 104 kbp) 04:24 07:48 02:06 08:52 06:28 01:50 02:29 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 10:39 10:23 09:26 02:22 07:00 07:34 03:46 02:42 02:10 02:07 07:41 01:49 01:44 01:39 01:33 02:40 02:20 11:59 01:42 02:13 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 08:49 12:14 09:31 07:31 11:02 09:40 06:41 08:36 08:21 04:47 05:14 03:15 03:06 02:11 02:07 02:09 02:53 01:52 01:43 01:30 01:55 01:24 02:05 01:44 01:43 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 01:44 03:58 08:19 09:29 03:40 01:44 02:51 01:44 01:44 01:55 02:09 01:40 04:24 01:26 01:59 05:54 08:21 01:57 01:38 02:04 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 08:08 08:39 06:33 06:40 01:41 08:48 02:08 02:05 01:55 05:50 02:21 02:24 02:23 05:19 01:38 02:11 08:40 01:40 01:40 01:34 04:48 05:38 01:49 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 09:29 03:47 02:44 02:08 03:13 01:58 01:55 02:27 01:29 01:47 02:01 01:53 03:04 04:11 01:37 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 06:07 07:09 05:09 04:08 01:55 01:50 01:37 08:43 10:01 01:39 02:13 01:40 08:45 07:54 01:20 01:31 02:11 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 08:58 02:52 03:55 03:42 04:42 02:30 02:08 01:38 01:47 01:40 02:34 01:50 02:46 03:27 A A A A A A A A A A A B A A A A A A A A A A A B A A A A A A A A A A A B A A A A A A A A A A A B 08:40 09:57 05:26 04:03 02:07 02:11 01:45 01:43 01:43 02:01 01:41 01:40 01:27 01:56 01:37 10:21 05:25 01:36 01:33 02:05

Single-cell Illumina assemblies - >15 Ca. T. hypermnestrae single-cell genomes were ampliﬁed, no Ca. T. oneisti genome could be ampliﬁed - Illumina-assemblies have ≤ 53 % completeness - Long ONT reads are spanning low-coverage Illumina reads

Ca. Thiosymbion oneisti genome (symbiont MAG) - 4.3 Mbp genome, 59 % GC - 94 % completeness, 0.75 % contamination

- Full pathways for S-oxidation, denitrification, CO2-fixation, ... - 24 different transposase types (acc. to COGs); Several gene cassettes (integrons) with 29 attC sites - No conserved gene synteny around dnaAN operon dnaN dnaA - Taken together, this might point to large-scale genome arrangements J16 dnaN dnaA - Petersen et al. (2016). Chemosynthetic symbionts of marine invertebrate animals are capable of nitrogen fixation. M13 Nat Microbiol 2: 16195. hyp - Weber et al. (2019). A Bidimensional Segregation Mode Maintains Symbiont Chromosome Orientation toward Its Host. hyp dnaA Curr Biol 0. e-pub ahead of print, doi: 10.1016/j.cub.2019.07.064. N20 dnaN dnaA hyp bchE N06 dnaA holC pepA lptG I15 hyp mobB gyrB dnaN dnaA K16 0 1000 2000 3000 4000 5000 6000 7000 8000