The genome sequence of the common limpet, Patella vulgata (Linnaeus, 1758)

We present a genome assembly from an individual Patella vulgata (the common limpet; Mollusca; Gastropoda; Patellogastropoda; Patellidae). The genome sequence is 695.4 megabases in span. Most of the assembly is scaffolded into 9 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 14.93 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,378 protein coding genes.


Background
Patella vulgata was first described by Linnaeus in 1758, and unlike the rest of the Patellidae, its taxonomic status has remained remarkably stable.Patella vulgata occurs from the Algarve in Portugal to the Artic Circle in Norway (Southward et al., 1995).It is present in the Faeroe Islands, but absent from Iceland.It is most abundant on more exposed rocky shores, extending into sheltered fucoid dominated shores, where it becomes less abundant.On northern European shores it is the dominant limpet between the tide marks on sheltered to exposed shores.It gives way to Patella depressa on the mid and upper shores in southwest Britain and becomes increasingly rare further south in Europe.Lower down on exposed shores and in rockpools it gives way to Patella ulysipponensis.P. vulgata is a protandrous hermaphrodite, first maturing as a male and later in life, individuals usually become female (Orton et al., 1956), although some may remain male all their lives.In addition, some females may revert to being male (Le Quesne & Hawkins, 2006).There is evidence that sex change is density dependent: when large females were selectively removed from populations simulation human predation, not only did onset of sexual maturity to become males occur at smaller sizes, nut progression from males to females also occurred at smaller sizes (Borges et al., 2015).It is a simple brood autumn-winter spawner (Bowman, 1977;Orton et al., 1956).
P. vulgata has a larval life of 5 to 14 days, with a feeding veliger stage (Dodd, 1956;Hodgson et al., 2007).Grazing by P. vulgata prevents recruitment of algae in the intertidal on moderately exposed and exposed shores (Hawkins et al., 2019;Hawkins & Hartnoll, 1983), in particular, preventing stands of fucoid algae developing.This was dramatically demonstrated following excessive application of toxic dispersants to clean up the Torrey Canyon oil spill in 1967.Vast numbers of limpets were killed on shores from Trevone to the Lizard on the Cornish coastline (Southward & Southward, 1978), leading to massive blooms of green and subsequently fucoid algae.Recovery occurred in a series of damped oscillations on the shores suffering the most from dispersant application, taking up to 15 years to recover (Hawkins et al., 2017;Hawkins & Southward, 1992).
It can be considered a keystone species in the Northeast Atlantic.In recent years recruitment failure has been noted in southwest England (Moore et al., 2011) and it has decreased in relative abundance to P. depressa on the mid and upper zones of moderately exposed and exposed shores in response to climate warming (Hawkins et al., 2008;Hawkins et al., 2009).

Genome sequence report
The genome was sequenced from a Patella vulgata specimen (Figure 1) collected from Godrevy, Cornwall, UK (50.24, -5.40).A total of 62-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 24-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 5 missing joins or mis-joins and removed one haplotypic duplication, reducing the scaffold number by 11.54%.
The final assembly has a total length of 695.4 Mb in 22 sequence scaffolds with a scaffold N50 of 87.4 Mb (Table 1).Most (99.96%) of the assembly sequence was assigned to 9 chromosomallevel 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 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.

Sample acquisition and nucleic acid extraction
A Patella vulgata (specimen ID MBA-200706-003A, individual xgPatVulg1) was collected from Godrevy, Cornwall, UK (latitude 50.24, longitude -5.40) on 2020-07-06.The specimen was collected by hand by Nova Mieszkowska  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.
RNA was extracted from muscle tissue of xgPatVulg1 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes (Garrison & Marth, 2012).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 using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021).Manual curation was performed using gEVAL, 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 (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Patella vulgata assembly (GCA_932274485.2) in Ensembl Rapid Release.

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 Is the rationale for creating the dataset(s) clearly described?
Yes.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 a keystone intertidal species that is very widespread and a key "engineer" of these ecosystems.

Are the protocols appropriate and is the work technically sound?
Yes, these various protocols are certainly appropriate and the work is of a high technical standard.

Are sufficient details of methods and materials provided to allow replication by others?
Yes, these are ell-described and meet accepted standards for these data notes.

Are the datasets clearly presented in a useable and accessible format?
Yes, the datasets such as the assembly and sequencing data are accessible and useable.
Other minor corrections.
The draft genome of Patella vulgata has been previously sequenced and published (Kenny et al., Marine Genomics 24 (2015), 139-146).I was surprised that this previous publication was not mentioned, nor any attempts made to put the current assembly into the context of this previous work. 1.
In the background section the sentence starting "There is evidence that sex change is density dependent…" does not make sense and needs rewriting.

2.
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: 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. .Also, I suggest passing the last sentence of the second paragraph to the beginning of the third paragraph: Such as most species of patellogastropod limpets, P. vulgata is a broadcast spawners,with an autumn-winter spawning period (Bowman, 1977;Orton et al., 1956).Its life cycle includes a pelagic larval phase (with a feeding veliger stage) that last between 5 and 14 days.Finally, I think that the final sentences of this paragraph ("This was dramatically…15 years to recover) are really anecdotic and digressive, and I suggest omitting these.

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: Evolutionary biology, developmental biology, comparative genomics 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 Patella vulgata, xgPatVulg1.2: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 695,382,709 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 (95,737,551 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (87,393,929 and 54,963,775 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/xgPatVulg1.2/dataset/CAKNZN02/snail.

Figure 3 .
Figure 3. Genome assembly of Patella vulgata, xgPatVulg1.2: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/xgPatVulg1.2/dataset/CAKNZN02/blob.

Figure 4 .
Figure 4. Genome assembly of Patella vulgata, xgPatVulg1.2: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/xgPatVulg1.2/dataset/CAKNZN02/cumulative.

Figure 5 .
Figure 5. Genome assembly of Patella vulgata, xgPatVulg1.2:Hi-C contact map of the xgPatVulg1.2assembly, 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=Bn4_scjASu-Z8fV43PKCaw.

Reviewer
Report 21 November 2023 https://doi.org/10.21956/wellcomeopenres.22154.r69414© 2023 Zardoya R.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.Rafael Zardoya Departamento de Biodiversidad y Biología Evolutiva, Museo Nacional de Ciencias Naturales (MNCN-CSIC), Madrid, Spain This genome report presents the sequencing, assembling and scaffolding of the 9-chromosome genome of the common limpet.This species is ecologically relevant in the rocky shores of Western Europe and belongs to the earliest divergent lineage in the Gastropoda.The pipeline from the initial sampling of the individual to the final scaffolding and preliminary annotation of the (almost) one-phased genome followed the high standards established by the Wellcome Sanger Institute.As a result, almost no signal of contamination is found and the BUSCO scores are correct for a gastropod.The comparison of this genome with those of Patella pelucida (Lawniczak et al. 2022) 1 and Lottia gigantea (Simakov et al. 2013) 2 will be particularly interesting.With regards to the introduction, I find useful citing that the mitogenome of P. vulgata was already sequenced (Uribe et al. 2019) 3