Genome sequence for the thick topshell, Phorcus lineatus (da Costa, 1778)

We present a genome assembly from an individual Phorcus lineatus (the thick topshell; Mollusca; Gastropoda; Trochida; Trochidae). The genome sequence is 958 megabases in span. Most of the assembly (99.9%) is scaffolded into 18 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 19.1 kilobases in length.


Background
The prosobranch gastropod Phorcus lineatus (da Costa, 1778), commonly called the thick topshell or toothed topshell, is a species of trochid commonly found on moderately exposed rocky shores in the south and west of the UK.It has a geographic distribution extending from Morocco (Lewis, 1996) to north Wales (Mieszkowska & Sugden, 2022).P. lineatus occurs among boulders, cobbles, and bedrock in the high and midshore zones of rocky intertidal habitats.Some seasonal migration downshore in the winter has been previously recorded (Crothers, 2001), but with climate change this has not been evident in UK populations studied between the 2000s and 2020s (MarClim unpublished data).
P. lineatus is one of the most abundant species of grazer occurring on rocky shores in the north-east Atlantic, and feeds off biofilm on rock surfaces (Desai, 1966).P. lineatus is a broadcast spawner with a lecithotrophic larval stage lasting for a few days (Desai, 1966).Recruits settle within the same intertidal habitat as the adults, but in cryptic habitats (Crothers, 2001) under boulders, cobbles, and in crevices.Maturation occurs at around two years of age (Garwood & Kendall, 1985), and individuals can live in excess of 20 years (Mieszkowska et al., 2006).P. lineatus is distinguished from other species of trochid by its black conical shell, often with an eroded apex exposing the nacreous shell layer.Growth checks are evident as vertical bands around the shell.There is a white columella projection near to the aperture which explains its common name.
This species has shown some of the fastest responses to climate change in any natural system, expanding the leading range edge in north Wales over 10 km per decade (Mieszkowska et al., 2006;Mieszkowska & Sugden, 2016).A high-quality genome sequence for this species will allow future studies to understand more about the mechanisms driving the observed response of this species to a changing climate.

Genome sequence report
The genome was sequenced from one Phorcus lineatus (Figure 1) collected from Godrevy in Cornwall, UK (latitude 50.24, longitude -5.396).A total of 32-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 51-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 225 missing joins or misjoins and removed 83 haplotypic duplications, reducing the assembly length by 3.87% and the scaffold number by 67.9% and increasing the scaffold N50 by 3.44%.
The final assembly has a total length of 958 Mb in 78 sequence scaffolds with a scaffold N50 of 49.8 Mb (Table 1).Most (99.9%) of the assembly sequence was assigned to 18 chromosomal-level scaffolds (Figure 2-Figure 5; Table 2).Chromosome 13 contains a possible heterozygous inversion in   the region of approximately 1-6.14 Mb.The orientation of the chromosome between these coordinates is not certain.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/1620919.  kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing.HMW DNA was sheared into an average fragment size between 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.

Sample acquisition and nucleic acid extraction
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 xgPhoLine2 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.(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.

Legal and ethical review process for Darwin Tree of Life Partner submitted materials
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.
The paper by Mieszkowska and Mrowicki combines data from PacBio and 10x to represent the genome of Phorcus lineatus in good quality to meet the Darwin Tree of Life criteria.By the Darwin Tree of Life's standard for publishing genomes, this genome data was shown to make the data widely available to various researchers.The report is commendable for covering the necessary and sufficient content for a genome report.
I have a slight concern about "manual curation" to keep reproducibility of the analysis.However, the other Darwin Tree of Life genome papers seem to have the same description and seem to be acceptable, so we hope that a detailed method paper will be published someday.

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: evolutionary biology and genome biology

I confirm that I have read this submission and believe that I have an appropriate level of
The assembly was generated using gold-standard technologies, i.e. a combination of PacBio HiFi reads, Illumina 10X and Hi-C libraries.These data allowed the assembly of a highly complete and contiguous assembly, which satisfied all VGP benchmarks, with the exception of BUSCO metrics.Nevertheless, the present version of the Mollusca dataset in OrthoDB is notoriously inappropriate for quality assessment in Mollusca, most likely due to the small number of high quality genomes used for its generation, thereby leading to significant underestimates of the actual quality of newly assembled molluscan genomes.The authors might therefore want to switch to a more reliable dataset for this assessment, such as the Metazoa ODB reference.
As in all other Darwin Tree of Life reports, the published genome currently lacks annotation, as clearly mentioned in the data availability statement.The authors briefly mentioned the presence of a single probable heterozygous inversion, but did not actually report any data about heterozygosity itself, which can be easily estimated from k-mer spectra analysis, even though k-mer representation in the assembly was very high.If this data is available, the authors could briefly mentioned whether the assembly size and inferred number of chromosomes are consistent with previous cytogenetic estimates.

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

Melody Clark
Natural Environment Research Council, Cambridge, England, UK This article describes the genome sequencing and assembly of the thick topshell, Phorcus lineatus via the Darwin Tree of Life programme.As would be expected of a genome produced by the Sanger Institute, it is produced using a very robust standard pipeline and is of the highest quality.
The background to the species is well described.The genome sequence report is produced using standard reporting metrics and displayed via standard bioinformatics packages.This gastropod genome is a useful addition to the expanding repertoire of mollusc genomes.

Rafael Zardoya
Departamento de Biodiversidad y Biología Evolutiva, Museo Nacional de Ciencias Naturales (MNCN-CSIC), Madrid, Spain The paper reports the sequencing at the chromosome level of the thick topshell genome-.The natural history of the species is well introduced and standard methods of sequencing, assembling and scaffolding are well explained.I have only few minor comments: the term "prosobranch" is outdated as it is from an old taxonomic classification of gastropods and represent a non-monophyletic group.I suggest using "vetigastropod" instead.
○ I think "in the south and west of the UK.It has a geographic distribution extending" can be deleted, as it is local compared to the whole distribution.

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The BUSCO value is relatively low.This is normal with many gastropods.Have the authors try using the metazoan odb, it sometimes increases the BUSCO value.
○ I know this is a report and must be short but it would be nice mentioning and comparing briefly this genome with that of the closely related Chrysomallon squamiferum ○ 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.
Reviewer Expertise: Gastropod phylogenomics and 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 Phorcus lineatus, xgPhoLine1.1: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 957,853,961 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 (79,430,111 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (49,790,511 and 44,639,602 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/xgPhoLine1.1/dataset/CAKLCT01.1/snail.
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing were performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq and 10X) instruments.Hi-C data were also generated from muscle tissue of xgPhoLine1 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).One round of polishing was

Figure 5 .
Figure 5. Genome assembly of Phorcus lineatus, xgPhoLine1.1:Hi-C contact map.Hi-C contact map of the xgPhoLine1.1 assembly, visualised using HiGlass.Chromosomes are given in order of size from left to right and top to bottom.An interactive version of the figure can be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=V15PjfA4QSC1MB2tiWmxzA.

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.

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.