The genome sequence of the Atlantic Strawberry Cockle, Americardia media (Linnaeus, 1758) (Cardiida: Cardiidae)
Ruiqi Li, Jingchun Li, Mark L. Blaxter, Rebekah L. Rogers, Kyle E McElroy
TL;DR
This paper presents the genome sequence of the Atlantic Strawberry Cockle, a bivalve mollusk, including two haplotypes and the mitochondrial genome.
Contribution
The study provides a high-quality genome assembly for Americardia media, including chromosomal pseudomolecules and a mitochondrial genome.
Findings
The genome assembly includes two haplotypes with total lengths of 1,299.87 and 1,284.99 megabases.
Haplotype 1 is scaffolded into 19 chromosomal pseudomolecules covering 99.42% of the assembly.
The mitochondrial genome is assembled with a length of 47.2 kilobases.
Abstract
We present a genome assembly from an individual Americardia media (Atlantic Strawberry Cockle; Mollusca; Bivalvia; Cardiida; Cardiidae). The assembly contains two haplotypes with total lengths of 1 299.87 megabases and 1 284.99 megabases. Most of haplotype 1 (99.42%) is scaffolded into 19 chromosomal pseudomolecules. Haplotype 2 was assembled to scaffold level. The mitochondrial genome has also been assembled, with a length of 47.2 kilobases.
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Figure 1
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Figure 6| Platform | PacBio HiFi | Hi-C |
|---|---|---|
|
| xbAmeMedi1 | xbAmeMedi1 |
|
| SAN20002327 | SAN20002327 |
|
| SAMEA115336917 | SAMEA115336917 |
|
| SAMEA115336924 | SAMEA115336924 |
|
| Whole organism | Whole organism |
|
| Revio | Illumina NovaSeq X |
|
| ERR13762720 | ERR13766898 |
|
| 6.66 million | 747.19 million |
|
| 67.68 Gb | 112.83 Gb |
|
| xbAmeMedi1.hap1.1 | xbAmeMedi1.hap2.1 |
|
| GCA_964300355.1 | GCA_964300375.1 |
|
| chromosome | scaffold |
|
| 1 299.87 | 1 284.99 |
|
| 19 | Scaffold-level |
|
| 632 | 507 |
|
| 5.6 Mb | 5.99 Mb |
|
| 267 | 174 |
|
| 67.44 Mb | 66.77 Mb |
|
| 105.39 | - |
|
| Mitochondrion: 47.2 kb | - |
| INSDC
| Molecule | Length
| GC% |
|---|---|---|---|
| 1 | 105.39 | 36 | |
| 2 | 103.60 | 36 | |
| 3 | 95.37 | 36 | |
| 4 | 89.52 | 36.50 | |
| 5 | 78.89 | 36.50 | |
| 6 | 76.43 | 36 | |
| 7 | 69.81 | 36 | |
| 8 | 67.44 | 36 | |
| 9 | 65.20 | 36.50 | |
| 10 | 64.76 | 36.50 | |
| 11 | 64.19 | 36.50 | |
| 12 | 64.10 | 36 | |
| 13 | 55.83 | 36.50 | |
| 14 | 55.18 | 36.50 | |
| 15 | 54.49 | 37 | |
| 16 | 49.71 | 36.50 | |
| 17 | 46.47 | 36.50 | |
| 18 | 43.02 | 36.50 | |
| 19 | 42.96 | 36.50 |
| Measure | Value | Benchmark |
|---|---|---|
| EBP summary (haplotype 1) | 6.C.Q65 | 6.C.Q40 |
| Contig N50 length | 5.60 Mb | ≥ 1 Mb |
| Scaffold N50 length | 67.44 Mb | = chromosome N50 |
| Consensus quality (QV) | Haplotype 1: 65.9; haplotype 2: 66.7; combined: 66.3 | ≥ 40 |
|
| Haplotype 1: 68.22%; Haplotype 2: 67.98%; combined: 98.90% | ≥ 95% |
| BUSCO | C:79.5% [S:78.5%; D:1.0%]; F:5.3%; M:15.1%; n:5 295 | S > 90%; D < 5% |
| Percentage of assembly
| 99.42% | ≥ 90% |
- —Wellcome Trust
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Taxonomy
TopicsGenomics and Phylogenetic Studies · Marine Biology and Ecology Research · Protist diversity and phylogeny
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Spiralia; Lophotrochozoa; Mollusca; Bivalvia; Autobranchia; Heteroconchia; Euheterodonta; Imparidentia; Neoheterodontei; Cardiida; Cardioidea; Cardiidae; Fraginae; Americardia; Americardia media (Linnaeus, 1758) (NCBI:txid241156)
Background
Americardia media is a widespread and morphologically variable cockle of the Caribbean and western Atlantic. Its range extends from southern Florida and the Gulf of Mexico southward to Panama, Suriname, and throughout the West Indies ( ter Poorten, 2024). This species inhabits sandy bottoms in shallow, sheltered environments, typically in intertidal and subtidal zones associated with seagrass beds or algal meadows ( ter Poorten, 2024).
Americardia media belongs to the subfamily Fraginae (heart cockles), which includes both a non-symbiotic lineage and a symbiotic lineage that maintains photosymbiotic relationships with dinoflagellates of the family Symbiodiniaceae ( Kirkendale, 2009; Li et al., 2020). This diversity makes Fraginae a powerful system for comparative studies on the origins and molecular mechanisms of animal photosymbiosis. While several genomes from photosymbiotic fraginids are already available ( Li et al., 2024a; Li et al., 2024b; Li et al., 2024c), the genome of A. media adds an essential non-symbiotic counterpart, enabling investigations into morphological adaptation to photosymbiosis and the divergent evolutionary trajectories of symbiotic versus non-symbiotic bivalves.
We present a chromosome-level genome sequence for Americardia media, the first genome for the genus Americardia and one of 14 genomes available for the family Cardiidae 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 West Palm Beach, Florida, Usa ( Figure 1)
Photograph of the Americardia media (xbAmeMedi1) specimen used for genome sequencing.
Methods
Sample acquisition
The specimen used for genome sequencing was an adult Americardia media (specimen ID SAN20002327, ToLID xbAmeMedi1; Figure 1), collected from West Palm Beach, Florida, USA (latitude 26.783, longitude –80.043) on 2022-06-01. The specimen was collected and identified by Ruiqi Li (University of Colorado, Boulder). The voucher id is UCM51385, deposited in the University of Colorado Boulder Museum of Natural History.
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 xbAmeMedi1 sample was weighed and triaged to determine the appropriate extraction protocol. Tissue 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 4.4 ng/μL and a yield of 572.00 ng.
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 on a Revio instrument (Pacific Biosciences). The prepared library was normalised to 2 nM, and 15 μL was used for making complexes. Primers were annealed and polymerases bound to generate circularised complexes, following the manufacturer’s instructions. Complexes were purified using 1.2X SMRTbell beads, then diluted to the Revio loading concentration (200–300 pM) and spiked with a Revio sequencing internal control. The sample was sequenced on a Revio 25M SMRT cell. The SMRT Link software (Pacific Biosciences), a web-based workflow manager, was used to configure and monitor the run and to carry out primary and secondary data analysis.
Hi-C
** Sample preparation and crosslinking **
The Hi-C sample was prepared from 20–50 mg of frozen tissue of the xbAmeMedi1 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 to 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 X.
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 in Hi-C phasing mode ( Cheng et al., 2021; Cheng et al., 2022), producing two haplotypes. Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019). Contigs were further scaffolded with Hi-C data in YaHS ( Zhou et al., 2023), using 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. The mitochondrial genome was assembled using OATK ( Zhou et al., 2025).
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 21 breaks and 44 joins. This reduced the scaffold count by 49.6% and reduced the total assembly length by 1.8%. The curation process is described 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 both 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 Americardia media specimen generated 67.68 Gb (gigabases) from 6.66 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 1 251.78 Mb, with a heterozygosity of 2.12% and repeat content of 42.88% ( Figure 2). These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 52× coverage. Hi-C sequencing produced 112.83 Gb from 747.19 million reads, which were used to scaffold the assembly. Table 1 summarises the specimen and sequencing details.
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.
Assembly statistics
The genome was assembled into two haplotypes using Hi-C phasing. Haplotype 1 was curated to chromosome level, while haplotype 2 was assembled to scaffold level. The final assembly has a total length of 1 299.87 Mb in 267 scaffolds, with 365 gaps, and a scaffold N50 of 67.44 Mb ( Table 2).
Most of the assembly sequence (99.42%) was assigned to 19 chromosomal-level scaffolds. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 3; Table 3). During curation, we observed haplotypic inversions in the following regions: chromosome 3 (4.6–53.7 Mbp) and 5 (21.3–51.2 Mbp).
Hi-C contact map of the Americardia media 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 haplotype 1 genome assembly of Americardia media xbAmeMedi1.
The mitochondrial genome was also assembled (length 47.2 kb, OZ199140.1). This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.
For haplotype 1, the estimated QV is 65.9, and for haplotype 2, 66.7. When the two haplotypes are combined, the assembly achieves an estimated QV of 66.3. The k-mer completeness is 68.22% for haplotype 1, 67.98% for haplotype 2, and 98.90% for the combined haplotypes ( 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 analysis using the mollusca_odb10 reference set ( n = 5 295) identified 79.5% of the expected gene set (single = 78.5%, duplicated = 1.0%) for haplotype 1. The snail plot in Figure 5 summarises the scaffold length distribution and other assembly statistics for haplotype 1. The blob plot in Figure 6 shows the distribution of scaffolds by GC proportion and coverage for haplotype 1.
Assembly metrics for xbAmeMedi1.hap1.1.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 set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer.
BlobToolKit GC-coverage plot for xbAmeMedi1.hap1.1.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 haplotype 1, is 6.C.Q65, meeting the recommended reference standard.
Table 4.: Earth Biogenome Project summary metrics for the Americardia media assembly.
Wellcome Sanger Institute – Legal and Governance
The materials that have contributed to this genome note have been supplied by a Tree of Life collaborator. 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 materialLegality of collection, transfer and use (national and international)
Each transfer of samples is undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Tree of Life collaborator, Genome Research Limited (operating as the Wellcome Sanger Institute) and in some circumstances other Tree of Life collaborators.
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