Abstract
We present a genome assembly from an individual Cepaea hortensis (white-lipped garden snail; Mollusca; Gastropoda; Stylommatophora; Helicidae). The genome sequence has a total length of 3 168.99 megabases. Most of the assembly (97.52%) is scaffolded into 22 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, with a length of 15.08 kilobases. Gene annotation of this assembly on Ensembl identified 17 974 protein-coding genes. This assembly was generated as part of the Darwin Tree of Life project, which produces reference genomes for eukaryotic species found in Britain and Ireland.
Keywords: Cepaea hortensis; white-lipped garden snail; genome sequence; chromosomal; Stylommatophora
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Spiralia; Lophotrochozoa; Mollusca; Gastropoda; Heterobranchia; Euthyneura; Panpulmonata; Eupulmonata; Stylommatophora; Helicina; Helicoidea; Helicidae; Cepaea; Cepaea hortensis (Müller, 1774) (NCBI:txid97200)
Background
Cepaea hortensis (Müller, 1774), commonly known as the white-lipped snail, is a terrestrial pulmonate gastropod in the family Helicidae. It is widely distributed across northern and central Europe, with a more northerly range than its sister species C. nemoralis, extending into northern Scandinavia and Iceland. C. hortensis has also been present in northeastern coastal regions of North America for several thousand years, as evidenced by archaeological deposits ( Pearce et al., 2010).
Although less extensively studied than C. nemoralis, C. hortensis shares many of the same features that together make the genus Cepaea a valuable model in evolutionary biology and ecological genetics. Like its sister species, C. hortensis exhibits a striking shell colour and banding polymorphism, although the degree of variation within and between populations is less.
In most regions, the shell of C. hortensis can be distinguished from C. nemoralis by a pale or white lip around the shell aperture in adults, whereas C. nemoralis typically has a brown or black lip. However, this distinction is not universally reliable: in some areas, such as Ireland and the Pyrenees, C. nemoralis may have a white lip, and C. hortensis can occasionally have a dark lip ( Cameron, 1969; Cameron et al., 1973; Ramos-Gonzalez, 2021) Shell size also tends to differ, with C. hortensis generally smaller than C. nemoralis, though there is considerable overlap. Where the two species co-occur, character displacement may occur, but this is not consistent across all populations.
Comparative studies between C. hortensis and C. nemoralis have been valuable in understanding the evolutionary dynamics of shell polymorphism ( Carter, 1968; Clarke, 1962). While C. hortensis has been less frequently used in experimental and field studies, it provides an important contrast in terms of habitat preference, geographic distribution, and population structure. Its presence in cooler and more northerly environments offers insights into climatic selection and the maintenance of polymorphism under different ecological pressures.
We present a chromosome‑level genome sequence for Cepaea hortensis, one of two new genomes for the genus Cepaea as of September 2025 (data obtained via NCBI datasets, O’Leary et al., 2024). The assembly was produced using the Tree of Life pipeline from a specimen collected in Geddington, Kettering, Northamptonshire, UK ( Figure 1). This assembly was generated as part of the Darwin Tree of Life Project, which aims to generate high‑quality reference genomes for all named eukaryotic species in Britain and Ireland to support research, conservation, and the sustainable use of biodiversity ( Darwin Tree of Life Project Consortium, 2022).
Figure 1. Photograph of the shell of the Cepaea hortensis (xgCepHort1) specimen used for genome sequencing.
Methods
Sample acquisition
The specimens used for sequencing were adult Cepaea hortensis collected from a private garden in Geddington, Kettering, Northamptonshire, United Kingdom (latitude 52.4391, longitude –0.6831) on 2019-12-05. The genome was sequenced from one specimen using PacBio HiFi technology (specimen ID SAN0001318, ToLID xgCepHort1; Figure 1). A second specimen was used for Hi-C sequencing (specimen ID SAN0001319, ToLID xgCepHort2), and a third for RNA sequencing (specimen ID SAN0001321, ToLID xgCepHort4). The specimens were collected by Chris Wade and identified by Angus Davison. For the Darwin Tree of Life sampling and metadata approach, refer to Lawniczak et al. (2022).
Nucleic acid extraction
Protocols for high molecular weight (HMW) DNA extraction developed at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory are available on protocols.io ( Howard et al., 2025). The xgCepHort1 sample was weighed and triaged to determine the appropriate extraction protocol. Tissue from the head was homogenised by powermashing using a PowerMasher II tissue disruptor.
HMW DNA was extracted using the Automated MagAttract v2 protocol. DNA was sheared into an average fragment size of 12–20 kb following the Megaruptor®3 for LI PacBio protocol. Sheared DNA was purified by automated SPRI (solid-phase reversible immobilisation). The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system. For this sample, the final post-shearing DNA had a Qubit concentration of 15.7 ng/μL and a yield of 2 041.00 ng. The 260/280 spectrophotometric ratio was 1.97, and the 260/230 ratio was 2.37.
RNA was also extracted from posterior body tissue of xgCepHort4 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol. The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit. Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
PacBio HiFi library preparation and sequencing
Library preparation and sequencing were performed at the WSI Scientific Operations core. Libraries were prepared using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, California, USA), following the manufacturer’s instructions. The kit includes reagents for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead clean-up, and nuclease treatment. Size selection and clean-up were performed using diluted AMPure PB beads (Pacific Biosciences). DNA concentration was quantified using a Qubit Fluorometer v4.0 (ThermoFisher Scientific) and the Qubit 1X dsDNA HS assay kit. Final library fragment size was assessed with the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) using the gDNA 55 kb BAC analysis kit.
The sample was sequenced using the Sequel IIe system (Pacific Biosciences, California, USA). The concentration of the library loaded onto the Sequel IIe was in the range 40–135 pM. The SMRT link software, a PacBio web-based end-to-end workflow manager, was used to set-up and monitor the run, and to perform primary and secondary analysis of the data upon completion.
Hi-C
Sample preparation and crosslinking
The Hi-C sample was prepared from 20–50 mg of frozen head tissue from the xgCepHort2 sample using the Arima-HiC v2 kit (Arima Genomics). Following the manufacturer’s instructions, tissue was fixed and DNA crosslinked using TC buffer to a final formaldehyde concentration of 2%. The tissue was homogenised using the Diagnocine Power Masher-II. Crosslinked DNA was digested with a restriction enzyme master mix, biotinylated, and ligated. Clean-up was performed with SPRISelect beads before library preparation. DNA concentration was measured with the Qubit Fluorometer (Thermo Fisher Scientific) and Qubit HS Assay Kit. The biotinylation percentage was estimated using the Arima-HiC v2 QC beads.
Hi-C library preparation and sequencing
Biotinylated DNA constructs were fragmented using a Covaris E220 sonicator and size selected to 400–600 bp using SPRISelect beads. DNA was enriched with Arima-HiC v2 kit Enrichment beads. End repair, A-tailing, and adapter ligation were carried out with the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs), following a modified protocol where library preparation occurs while DNA remains bound to the Enrichment beads. Library amplification was performed using KAPA HiFi HotStart mix and a custom Unique Dual Index (UDI) barcode set (Integrated DNA Technologies). Depending on sample concentration and biotinylation percentage determined at the crosslinking stage, libraries were amplified with 10–16 PCR cycles. Post-PCR clean-up was performed with SPRISelect beads. Libraries were quantified using the AccuClear Ultra High Sensitivity dsDNA Standards Assay Kit (Biotium) and a FLUOstar Omega plate reader (BMG Labtech).
Prior to sequencing, libraries were normalised to 10 ng/μL. Normalised libraries were quantified again and equimolar and/or weighted 2.8 nM pools were created. Pool concentrations were checked using the Agilent 4200 TapeStation (Agilent) with High Sensitivity D500 reagents before sequencing. Sequencing was performed using paired-end 150 bp reads on the Illumina NovaSeq 6000.
RNA library preparation and sequencing
Libraries were prepared using the NEBNext ® Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer’s instructions. Poly(A) mRNA in the total RNA solution was isolated using oligo(dT) beads, converted to cDNA, and uniquely indexed; 14 PCR cycles were performed. Libraries were size-selected to produce fragments between 100–300 bp. Libraries were quantified, normalised, pooled to a final concentration of 2.8 nM, and diluted to 150 pM for loading. Sequencing was carried out on the Illumina NovaSeq 6000 to generate 150-bp paired-end reads.
Genome assembly
Prior to assembly of the PacBio HiFi reads, a database of k-mer counts ( k = 31) was generated from the filtered reads using FastK. GenomeScope2 ( Ranallo-Benavidez et al., 2020) was used to analyse the k-mer frequency distributions, providing estimates of genome size, heterozygosity, and repeat content.
The HiFi reads were assembled using Hifiasm ( Cheng et al., 2021) with the --primary option. Haplotypic duplications were identified and removed using purge_dups ( Guan et al., 2020). The Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019), and the contigs were scaffolded in YaHS ( Zhou et al., 2023) with the --break option for handling potential misassemblies. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al., 2022), BUSCO ( Manni et al., 2021) and MERQURY.FK ( Rhie et al., 2020).
The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Assembly curation
The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants ( ASCC) pipeline. TreeVal was used to generate the flat files and maps for use in curation. Manual curation was conducted primarily in PretextView and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Manual corrections included 71 breaks, 116 joins, and removal of 33 haplotypic duplications. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation. PretextSnapshot was used to generate a Hi-C contact map of the final assembly.
Assembly quality assessment
The Merqury.FK tool ( Rhie et al., 2020) was run in a Singularity container ( Kurtzer et al., 2017) to evaluate k-mer completeness and assembly quality for the primary and alternate haplotypes using the k-mer databases ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.
The genome was analysed using the BlobToolKit pipeline, a Nextflow implementation of the earlier Snakemake version ( Challis et al., 2020). The pipeline aligns PacBio reads using minimap2 ( Li, 2018) and SAMtools ( Danecek et al., 2021) to generate coverage tracks. It runs BUSCO ( Manni et al., 2021) using lineages identified from the NCBI Taxonomy ( Schoch et al., 2020). For the three domain-level lineages, BUSCO genes are aligned to the UniProt Reference Proteomes database ( Bateman et al., 2023) using DIAMOND blastp ( Buchfink et al., 2021). The genome is divided into chunks based on the density of BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database with DIAMOND blastx. Sequences without hits are chunked using seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). The BlobToolKit suite consolidates all outputs into a blobdir for visualisation. The BlobToolKit pipeline was developed using nf-core tooling ( Ewels et al., 2020) and MultiQC ( Ewels et al., 2016), with containerisation through Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017).
Genome sequence report
Sequence data
PacBio sequencing of the Cepaea hortensis specimen generated 233.46 Gb (gigabases) from 20.66 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 2 869.57 Mb, with a heterozygosity of 1.28% and repeat content of 56.15% ( Figure 2). These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 45× coverage. Hi-C sequencing produced 357.04 Gb from 2 364.53 million reads, which were used to scaffold the assembly. RNA sequencing data were also generated and are available in public sequence repositories. Table 1 summarises the specimen and sequencing details.
Figure 2. Frequency distribution of k-mers generated using GenomeScope2.
The plot shows observed and modelled k-mer spectra, providing estimates of genome size, heterozygosity, and repeat content based on unassembled sequencing reads.
Table 1. Specimen and sequencing data for BioProject PRJEB70264.
| Platform | PacBio HiFi | Hi-C | RNA-seq |
|---|---|---|---|
| ToLID | xgCepHort1 | xgCepHort2 | xgCepHort4 |
| Specimen ID | SAN0001318 | SAN0001319 | SAN0001321 |
| BioSample (source individual) | SAMEA9312581 | SAMEA9312582 | SAMEA9312584 |
| BioSample (tissue) | SAMEA9312651 | SAMEA9312659 | SAMEA9312674 |
| Tissue | head | head | posterior body |
| Instrument | Sequel IIe | Illumina NovaSeq 6000 | Illumina NovaSeq 6000 |
| Run accessions | ERR12319372; ERR12319373; ERR12319369; ERR12319368; ERR12319370; ERR12319371 | ERR12318600 | ERR12318601 |
| Read count total | 20.66 million | 2 364.53 million | 43.63 million |
| Base count total | 233.46 Gb | 357.04 Gb | 6.59 Gb |
Assembly statistics
The primary haplotype was assembled, and contigs corresponding to an alternate haplotype were also deposited in INSDC databases. The final assembly has a total length of 3 168.99 Mb in 728 scaffolds, with 4 264 gaps, and a scaffold N50 of 127.28 Mb ( Table 2).
Table 2. Genome assembly statistics.
| Assembly name | xgCepHort1.1 |
| Assembly accession | GCA_963921405.1 |
| Alternate haplotype accession | GCA_963921395.1 |
| Assembly level | chromosome |
| Span (Mb) | 3 168.99 |
| Number of chromosomes | 22 |
| Number of contigs | 4 992 |
| Contig N50 | 1.21 Mb |
| Number of scaffolds | 728 |
| Scaffold N50 | 127.28 Mb |
| Organelles | Mitochondrion: 15.08 kb |
Most of the assembly sequence (97.52%) was assigned to 22 chromosomal-level scaffolds. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 3; Table 3).
Figure 3. Hi-C contact map of the Cepaea hortensis genome assembly.
Assembled chromosomes are shown in order of size and labelled along the axes, with a megabase scale shown below. The plot was generated using PretextSnapshot.
Table 3. Chromosomal pseudomolecules in the primary genome assembly of Cepaea hortensis xgCepHort1.
| INSDC accession | Molecule | Length (Mb) | GC% |
|---|---|---|---|
| OY992876.1 | 1 | 530.13 | 41.50 |
| OY992877.1 | 2 | 221.73 | 41 |
| OY992878.1 | 3 | 172.21 | 41 |
| OY992879.1 | 4 | 165.41 | 41.50 |
| OY992880.1 | 5 | 144.54 | 41 |
| OY992881.1 | 6 | 134.71 | 41 |
| OY992882.1 | 7 | 129.81 | 42 |
| OY992883.1 | 8 | 127.28 | 41.50 |
| OY992884.1 | 9 | 124.30 | 41.50 |
| OY992885.1 | 10 | 117.03 | 41.50 |
| OY992886.1 | 11 | 116.85 | 41.50 |
| OY992887.1 | 12 | 114.40 | 41 |
| OY992888.1 | 13 | 108.22 | 41.50 |
| OY992889.1 | 14 | 106.38 | 41 |
| OY992890.1 | 15 | 105.34 | 41.50 |
| OY992891.1 | 16 | 103.69 | 41.50 |
| OY992892.1 | 17 | 103.38 | 41.50 |
| OY992893.1 | 18 | 100.04 | 41.50 |
| OY992894.1 | 19 | 99.27 | 41.50 |
| OY992895.1 | 20 | 91.61 | 41.50 |
| OY992896.1 | 21 | 90.95 | 41.50 |
| OY992897.1 | 22 | 83.21 | 41.50 |
The mitochondrial genome was also assembled. This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.
The combined primary and alternate assemblies achieve an estimated QV of 53.8. The k-mer completeness is 82.34% for the primary assembly, 71.01% for the alternate haplotype, and 97.62% for the combined assemblies ( Figure 4).
Figure 4. Evaluation of k-mer completeness using MerquryFK.
This plot illustrates the recovery of k-mers from the original read data in the final assemblies. The horizontal axis represents k-mer multiplicity, and the vertical axis shows the number of k-mers. The black curve represents k-mers that appear in the reads but are not assembled. The green curve corresponds to k-mers shared by both haplotypes, and the red and blue curves show k-mers found only in one of the haplotypes.
BUSCO v.5.5.0 analysis of the primary assembly using the mollusca_odb10 reference set ( n = 5 295) identified 89.9% of the expected gene set (single = 71.4%, duplicated = 18.6%). The snail plot in Figure 5 summarises the scaffold length distribution and other assembly statistics for the primary assembly. The blob plot in Figure 6 shows the distribution of scaffolds by GC proportion and coverage.
Figure 5. Assembly metrics for xgCepHort1.1.
The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1 000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the mollusca_odb10 set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer.
Figure 6. BlobToolKit GC-coverage plot for xgCepHort1.1.
Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available on the BlobToolKit viewer.
Table 4 lists the assembly metric benchmarks adapted from Rhie et al. (2021) and the Earth BioGenome Project Report on Assembly Standards September 2024. The EBP metric, calculated for the primary assembly, is 6.C.Q53, meeting the recommended reference standard.
Table 4. Earth Biogenome Project summary metrics for the Cepaea hortensis assembly.
| Measure | Value | Benchmark |
|---|---|---|
| EBP summary (primary) | 6.C.Q53 | 6.C.Q40 |
| Contig N50 length | 1.21 Mb | ≥ 1 Mb |
| Scaffold N50 length | 127.28 Mb | = chromosome N50 |
| Consensus quality (QV) | Primary: 53.6; alternate: 53.9; combined: 53.8 | ≥ 40 |
| k-mer completeness | Primary: 82.34%; alternate: 71.01%; combined: 97.62% | ≥ 95% |
| BUSCO | C:89.9% [S:71.4%; D:18.6%]; F:2.4%; M:7.7%; n:5 295 | S > 90%; D < 5% |
| Percentage of assembly assigned to chromosomes | 97.52% | ≥ 90% |
Genome annotation report
The Cepaea hortensis genome assembly (GCA_963921405.1) was annotated by Ensembl at the European Bioinformatics Institute (EBI) using the Ensembl non-vertebrate genome annotation system. This annotation includes 61 358 transcribed mRNAs from 17 974 protein-coding and 34 600 non-coding genes. The average transcript length is 12 626.57 bp. There are an average of 1.17 coding transcripts per gene and 3.67 exons per transcript. For further information about the annotation, please refer to the annotation page on Ensembl.
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. 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.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (220540) and the Darwin Tree of Life Discretionary Award [218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328 </a>].
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 1; peer review: 2 approved]
Data availability
European Nucleotide Archive: Cepaea hortensis (white-lipped snail). Accession number PRJEB70264. The genome sequence is released openly for reuse. The Cepaea hortensis genome sequencing initiative is part of the Darwin Tree of Life Project (PRJEB40665) and the Sanger Institute Tree of Life Programme (PRJEB43745). All raw sequence data and the assembly have been deposited in INSDC databases. The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1 and Table 2.
Production code used in genome assembly at the WSI Tree of Life is available at https://github.com/sanger-tol. Table 5 lists software versions used in this study.
Table 5. Software versions and sources.
Author information
Contributors are listed at the following links:
Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team
Members of Wellcome Sanger Institute Scientific Operations – Sequencing Operations
Members of the Wellcome Sanger Institute Tree of Life Core Informatics team
Members of the Tree of Life Core Informatics collective
Members of the Darwin Tree of Life Consortium
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