The genome sequence of the Heterolobosean amoeboflagellate, Tetramitus jugosus CCAP 1588/3C

We present a genome assembly from cultivated Tetramitus jugosus (Heterolobosea; Schizopyrenida; Vahlkampfiidae). The genome sequence is 26.3 megabases in span. Most of the assembly (99.3%) is scaffolded into 52 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 49.46 kilobases in length.


Background
Tetramitus jugosus CCAP 1588/3C (Brown & de Jonckheere, 1999) is an Heterolobosean amoeboflagellate characterised by movement through pseudopodia, intranuclear promitosis, flattened mitochondrial cristae and an absence of stacked Golgi bodies (Page & Blanton, 1985).Tetramitus belongs to the family Vahlkampfiidae, and was originally established as a new genus, Paratretamitus, due to its capacity to transform into flagellated stages (Darbyshire et al., 1976), however, it was later transferred to the genus Tetramitus following the sequencing of its small subunit ribosomal DNA (SSU rDNA) (Brown & de Jonckheere, 1999;de Jonckheere et al., 2011).
T. jugosus can shift between an amoeboid vegetative state and flagellated form (Figure 1a), and, under unfavourable environmental conditions, it can form dormant cysts (Figure 1b) to survive in the environment.Once the conditions become favourable, they can excyst back to the ameboid form.Tetramitus feeds on bacteria, fungi, or other protists, engulfing them by phagocytosis into digestive vacuoles.
The Culture Collection of Algae and Protozoa T. jugosus strain (CCAP 1588/3C) was isolated in 1974 from soil collected at a depth of 5-15 cm in Morayshire, Scotland.The strain is maintained as a living culture and successfully cryopreserved at the CCAP, which ensures long-term safekeeping and open access to viable cultures of this organism.
Tetramitus are important members of soil microbial communities and predators of bacteria that play a key ecological role regulating microbial populations in the environment and therefore promoting plant growth (Bonkowski, 2004;Rosenberg et al., 2009).
The genome of T. jugosus CCAP 1588/3C will help address a grand challenge in protists research, namely the lack of relevant genome sequences of important protozoa species.

Genome sequence report
The genome was sequenced from a sample of cultivated cells of T. jugosus (paTetJugo1).A total of 37-fold coverage in Pacific Biosciences single-molecule HiFi long was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 7 missing or misjoins and removed 7 haplotypic duplications, reducing the assembly length by 7.91% and the scaffold number by 1.79%, and decreasing the scaffold N50 by 5.57%.
The final assembly has a total length of 26.3 Mb in 55 sequence scaffolds with a scaffold N50 of 0.5 Mb (Table 1).A summary of the assembly statistics is shown in Figure 2, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.3%) of the assembly sequence was assigned to 52 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/166959.

Sample acquisition and nucleic acid extraction
T. jugosus CCAP 1588/3C was cultivated in Non-Nutrient agar (Page, 1976) streaked with a non-pathogenic strain of Escherichia coli as food source.The strain was incubated at 20°C with no direct light ca. 5 µmol m -1 s -1 for a period of 12:12 h light:dark.Once all the E. coli was consumed, the biomass was harvested by washing the cells from the agar with sterile Page's Amoeba Saline Solution (Page, 1976;Page, 1988) and pelleted by centrifugation.The pellet was snap frozen in liquid nitrogen, the samples were then stored at -80°C and shipped on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute.The paTetJugo1 sample was weighed and some of the sample was set aside for Hi-C sequencing.The cells were cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.Fragment size analysis of 0.01-0.5 ng of DNA was then performed using an Agilent FemtoPulse.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to sequencing; a minimum of 50 ng DNA was submitted for sequencing.HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30.Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from a sample of paTetJugo1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
The assembly process included the following sequence of steps: Tiara 1.0.1 (Karlicki et al., 2022) was run with PacBio HiFi reads.Reads classified as prokaryotic by Tiara were removed and the remaining reads were assembled using Hifiasm (Cheng et al., 2021) with default settings.The Hifiasm contigs were checked for contaminants using Tiara 1.0.1 and BLAST against NCBI nt and nr databases (May 2021 versions).Mitochondrial and plastid contigs were detected using Tiara.The longest organellar contigs were circularised using circlator minimus2 (1.5.5) (Hunt et al., 2015).The remaining copies of organellar contigs were discarded.Deduplication of chromosomal contigs was done using GAP5 (1.2.A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were     et al., 2021;Simão et al., 2015) were calculated.
Table 3 contains a list of relevant software tool versions and sources.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material • Legality of collection, transfer and use (national and international) Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.

Open Peer Review
Current Peer Review Status: Oleg N. Shchepin University Greifswald, Greifswald, Germany The authors provide a high-quality assembly of the nuclear and mitochondrial genomes of Tetramitus jugosus.The resulting data are clearly described and are available from the ENA.These data will be valuable for further studies of the phylogeny and evolution of Heterolobosea.For the sake of reproducibility, it would be beneficial for the readers if the authors provided a more indepth description of the methods of genome assembly and curation, either in the form of the analysis script or specifying the parameter settings for the software they used.
Is the rationale for creating the dataset(s) clearly described?

Jeremy Wideman
Arizona State University, Arizona, USA The data note presents an excellent assembly of a heterolobosean, Tetramitus jugosus.The data appear solid and the methods robust.These data will be instrumental in future comparative analyses as they appear to represent 52 chromosome-length contigs.The methods and data provide a template and standard for future sequencing efforts in this clade.
Is the rationale for creating the dataset(s) clearly described?

Alexander K. Tice
Biological Sciences, Texas Tech University (Ringgold ID: 6177), Lubbock, Texas, USA The authors present the first nuclear and mitochondrial genome assemblies for the free-living heterolobsean amoeboflagellate Tetramitus jugosus.Through generation of long-read and conformational Hi-C sequence data the authors were able to produce a highly contiguous and complete nuclear genome along with a circularized mitochondrial genome.These data are a valuable contribution to the comparative genomics community as there are currently only a few publicly available genomes from heterolobosean taxa outside of the genus Naeglaria.The authors have made all the raw reads as well as the assemblies themselves publicly available, but the full annotation of the nuclear genome is still in progress.A few welcome additions/clarifications to the manuscript would be 1) any explanation as to the presence of the chromosome assigned to the phylum Arthropoda in the final assembly and 2) more detail on the specific flags and parameter choices in the many software packages used to produce the final assembly.
Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Partly
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: protistology, comparative genomics, phylogenetics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of T. jugosus, paTetJugo1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 26,350,207 bp assembly.The distribution of chromosome lengths is shown in dark grey with the plot radius scaled to the longest chromosome present in the assembly (699,783 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 chromosome lengths (499,551 and 399,693 bp), respectively.The pale grey spiral shows the cumulative chromosome count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the eukaryota_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/paTetJugo1.1/dataset/CALMHQ01/snail.

Figure 3 .
Figure 3. Genome assembly of T. jugosus, paTetJugo1.1:GC coverage.BlobToolKit GC-coverage plot.Chromosomes are coloured by phylum.Circles are sized in proportion to chromosome length.Histograms show the distribution of chromosome length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/paTetJugo1.1/dataset/CALMHQ01/blob.

Figure 5 .
Figure 5. Genome assembly of Tetramitus jugosus, paTetJugo1.1:Hi-C contact map of the paTetJugo1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=LpwbNNzHQsO6-sVUyCcfRQ.

Figure 4 .
Figure 4. Genome assembly of T. jugosus, paTetJugo1.1:cumulative sequence.BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all chromosomes.Coloured lines show cumulative lengths of chromosomes assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/paTetJugo1.1/dataset/ CALMHQ01/cumulative.

Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests
: No competing interests were disclosed.Reviewer Expertise: Comparative genomics, protistology, evolutionary cell biology I

confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
https://doi.org/10.21956/wellcomeopenres.22352.r77982© 2024 Tice A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.