The genome sequence of the Whirlpool Ramshorn snail, Anisus vortex (Linnaeus, 1758)

We present a genome assembly from an individual Anisus vortex (the Whirlpool Ramshorn snail; Mollusca; Gastropoda; Hygrophila; Lymnaeoidea; Planorbidae). The genome sequence is 869.5 megabases in span. Most of the assembly is scaffolded into 18 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 13.57 kilobases in length.


Background
Anisus vortex, better known as the Whirlpool Ramshorn snail, is distributed across Europe and Western Asiatic, reaching up to Finland in the Arctic circle (Kerney, 1999).It is commonly found in lowland areas in standing or slowmoving rivers, canals, lakes and drainage ditches however is not tolerant of habitats prone to desiccation (Kerney, 1999;Welter-Schultes, 2012).Anisus vortex is listed as a species of least concern in the current IUCN Red List data (Moorkens et al., 2011).
In the UK Anisus vortex is abundant in southern, eastern and central England as well as central Ireland.It is absent or only found in localised areas in the north of England and Scotland (Kerney, 1999;Rowson et al., 2021).Some older records from the most easterly and northern Scottish reports may be misidentifications for Anisus leucostoma (Kerney, 1999).
The shell is 7-10 mm in diameter, 1-2 mm in thickness and nearly opaque, yellow/brown in colour and is thin, smooth and glossy consisting of 6 to 7 whorls (Figure 1).The sharp keel is runs along the upper edge of the shell, whilst the underside is more rounded giving the aperture a more rhomboid like shape (Glöer, 2002;Rowson et al., 2021).The live animal is commonly dark purple-grey with white or colourless tentacles (Rowson et al., 2021).Juveniles are proportionally thicker in size but can be easily recognised by the strongly displaced keel (Rowson et al., 2021).
Anisus vortex is easily distinguished from other genera within the family by the sharp keel and angled aperture (Rowson et al., 2021).Anisus vortex can be easily confused with A. vorticulus, which is much rarer with more specific habitat requirements.These species can be separated by the more central keel in A. vorticulus and the more rhomboid profile of the shell in A. vortex (Kerney, 1999) or by dissection of the genital anatomy (Rowson et al., 2021).
Eggs are up to 0.5 mm in diameter and are laid in capsules, ovoid in shape and around 4 mm long, each containing 10 to 12 eggs (Rowson et al., 2021;Welter-Schultes, 2012).
Along with other planorbid species, Anisus vortex has been shown to be an intermediate parasite host to several species of vertebrate parasites including Alaria alata, which has been shown to infect domestic dogs as well as foxes (Portier et al., 2012).
The genome of Anisus vortex was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.Here we present a chromosomally complete genome sequence for Anisus vortex, based on one specimen from Pocklington Canal, York, UK.

Genome sequence report
The genome was sequenced from one Anisus vortex collected from Pocklington Canal, York, UK (53.89, -0.85).A total of 43-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 46 missing joins or misjoins and removed 15 haplotypic duplications, reducing the assembly length by 0.7%, and decreasing the scaffold N50 by 4.99%.
The final assembly has a total length of 869.5 Mb in 235 sequence scaffolds with a scaffold N50 of 45.4 Mb (Table 1).Most (96.27%) of the assembly sequence was assigned to 18 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotypehave 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/271030.

Sample acquisition and nucleic acid extraction
An Anisus vortex (specimen ID NHMUK014360733, individual xgAniVort1) was collected from Pocklington Canal, UK (latitude 53.89, longitude -0.85) on 2019-03-19 using a kicknet.The specimen was collected and identified by Sue Skipp (Environment Agency), and was then snap-frozen in a dry shipper.(Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.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    The rationale is clear.Besides the need for sequencing this mollusc as part of the Darwin Tree of Life project, there is the added interest of this animal being an intermediate host for a parasitic trematode that can infect mammals such as dogs and cats, but also potentially humans.

INSDC accession
The various protocols are certainly appropriate and the work is of a high technical standard.
The materials and methods descriptions meet accepted standards for these data notes, and the datasets such as the assemblies of the two haplotypes are accessible and useable.
There are a couple of minor typos for correction.Typo in Background, third paragraph: delete 'is' from "The sharp keel is runs along".1.
Genome sequence report section, second paragraph, line 8: insert a space to read, "haplotype have".

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.

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.
Reviewer Report 21 August 2023 https://doi.org/10.21956/wellcomeopenres.21968.r65479© 2023 Hui J.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.

Jerome H L Hui
The Chinese University of Hong Kong, Hong Kong, China Skipp and colleagues report the genome sequence of freshwater ramshorn snail Anisus vortex (Linnaeus 1758).This species can be found in most places in Britain.Molecular data of this species are mainly confined to COI and ribosomal sequences deposited to the NCBI database.Since this species has also been found to be intermediate host of parasitic diseases in dogs and foxes, this new genome resource is important and will be very useful for further studies, such as understanding and prevention of the transmission of diseases, as well as evolutionary and genomics questions related to molluscs more widely.This genome resource is excellent from the summary statistics, with high BUSCO numbers, high sequence continuity (scaffold N50), and majority of sequences contained on the 18 pseudochromosomes (plus mitochondrion).To sum up, this is a valuable contribution.

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? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: I have published with Peter Holland more than three years ago, and confirm that this potential conflict of interest did not affect my ability to write an objective and unbiased review of the article.
Reviewer Expertise: Genomics, evolution, invertebrates I confirm that I have read this submission and believe that I have an appropriate level of

Figure 2 .
Figure 2. Genome assembly of Anisus vortex, xgAniVort1.1: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 869,544,677 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (86,562,697 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (45,417,137 and 34,151,741 bp), respectively.The pale grey spiral shows the cumulative scaffold 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 mollusca_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Anisus%20vortex/dataset/CASBPS01/snail.

Figure 3 .
Figure 3. Genome assembly of Anisus vortex, xgAniVort1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Anisus%20vortex/dataset/CASBPS01/blob.

Figure 4 .
Figure 4. Genome assembly of Anisus vortex, xgAniVort1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Anisus%20vortex/dataset/CASBPS01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Anisus vortex, xgAniVort1.1:Hi-C contact map of the xgAniVort1.1 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=Sbo4O9XrQoWcCW4SkeAH4A.

Table 1 . Genome data for Anisus vortex, xgAniVort1.1. Project accession data
(Allio et al., 2020), 2023).1%,M:3.8%,n:5,295C≥95%*AssemblymetricbenchmarksareadaptedfromcolumnVGP-2020 of "Table1: Proposed standards and metrics for defining genome assembly quality" from(Rhie et al., 2021).The xgAniVort1 sample was prepared at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The sample was weighed and dissected on dry ice.Whole organism tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle.DNA was extracted at the Wellcome Sanger Institute (WSI) Scientific Operations core using the Qiagen MagAttract HMW DNA kit, according to the manufacturer's instructions.SequencingPacific 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 Pacific Biosciences SEQUEL II and SEQUEL IIe instruments.Hi-C data were also generated from whole organism tissue of xgAniVort3 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.Genome assembly, curation and evaluationAssembly was carried out withHifiasm (Cheng et al., 2021)and haplotypic duplication was identified and removed with purge_dups(Guan et al., 2020).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS(Zhou et al., 2023).The assembly was checked for contamination and corrected as described previously(Howe et al., 2021).Manual curation was performed using HiGlass(Kerpedjiev  et al., 2018) and Pretext (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi(Uliano-Silva et al., 2023), which runs MitoFinder(Allio et al., 2020)or MITOS

Table 3
contains a list of relevant software tool versions and sources.