The genome sequence of the variegated scallop, Mimachlamys varia (Linnaeus, 1758)

We present a genome assembly from an individual Mimachlamys varia (the variegated scallop; Mollusca; Bivalvia; Pectinida; Pectinidae). The genome sequence is 975.4 megabases in span. Most of the assembly is scaffolded into 19 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 21.78 kilobases in length.


Background
The variegated scallop, Mimachlamys varia, is an inequivalve bivalve which is oval in shape with pronounced anterior ears and 25 to 35 prominent ribs, which have raised spatulate spines that increase in size towards the margin (Hayward & Ryland, 2017).Adults can usually reach up to 60 mm and are very variable in colour, ranging from off-white to yellow, orange, brown and purple or a combination of these colours as demonstrated by the sequenced specimen (Figure 1).Under laboratory conditions, larvae prefer to settle in sheltered areas of low light and vertical or sloping surfaces, with additional substrate increasing settlement (De la Roche et al., 2009;Rodhouse & Burnell, 1979).Once metamorphosis has occurred, juveniles mature as males but may change their sex several times as adults (Hayward & Ryland, 2017).M. varia attaches to substrate using byssus threads (Duncan et al., 2016) and can be found in high numbers in association with Modiolus modiolus beds alongside ascidians, hydroids and other epifaunal species (JNCC, 2022).Indeed, the specimen collected for genome sequencing was found attached to the underside of a small oblong marina buoy, in amongst similar epifauna.
M. varia is common in inner shore habitats around Britain and Ireland, northern Europe, the Mediterranean and West Africa.In Europe, the main commercial species of scallop are Pecten maximus and Aquipecten opecularis; however, M. varia is also landed alongside A. opecularis in France and Spain (Duncan et al., 2016).M. varia is also regarded as a useful tool for biomonitoring of water quality in ports and harbours on the coast of France (Barbarin et al., 2022;Breitwieser et al., 2018;Breitwieser et al., 2020).
Various genetic studies of M. varia have been published (Arias et al., 2009b;Arias et al., 2009a;Beajmont & Beveridge, 1984;Breitwieser et al., 2018;Fernandez-Moreno et al., 2008), most notably the sequencing of its transcriptome (Viricel et al., 2018) and whole mitochondrial genome (Malkócs et al., 2022).Here we present the chromosomally complete genome sequence for M. varia, sequenced as part of the Darwin Tree of Life Project.It is hoped that this will provide further insight into the biology, ecology and evolution of M. varia and other pectinid bivalves.

Genome sequence report
The genome was sequenced from one Mimachlamys varia specimen (Figure 1) collected from Haslar Marina, Gosport, UK (50.79, -1.12).A total of 44-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 29 missing joins or mis-joins and removed 8 haplotypic duplications, reducing the assembly length by 0.56% and the scaffold number by 8.79%.
The final assembly has a total length of 975.4 Mb in 82 sequence scaffolds with a scaffold N50 of 50.7 Mb (Table 1).Most (99.76%) of the assembly sequence was assigned to 19 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 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.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/50417.

Sample acquisition and nucleic acid extraction
The Mimachlamys varia specimen selected for genome sequencing was individual xbMimVari1, collected from the Haslar Marina, Gosport, UK (latitude 50.79, longitude -1.12) on 2020-09-24.The specimen was picked by hand from the underside of a small buoy attached to the marina pontoon by Chris Fletcher and Mary E. Spencer Jones (both Natural History Museum).The specimen was identified by Chris Fletcher and tissue extracted from the adductor muscle was  Longest scaffold (Mb) 65.9 * Assembly metric benchmarks are adapted from column VGP-2020 of "Table 1: Proposed standards and metrics for defining genome assembly quality" from (Rhie et al., 2021).
** BUSCO scores based on the mollusca_odb10 BUSCO set using v5.preserved in liquid nitrogen.The remainder of the specimen was preserved in 80% ethanol and stored at the Natural History Museum, London.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The xbMimVari1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Muscle tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.HMW DNA was sheared into an average fragment size of 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.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 xbMimVari1 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.

Sequencing
Pacific Biosciences HiFi circular consensus 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 was performed by the Scientific Operations core

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).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS.
The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016)  A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This work was    et al., 2015) were calculated.
Table 3 contains a list of relevant software tool versions and sources.

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 align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

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 "Sample acquisition and nucleic acid extraction" section.For example, what endonuclease was used to fragment crosslinked DNA and how was the library quality assessed?Also, in this section I noticed "$HIC_TISSUE" -I think this might be remnant text from a manuscript template?
For the Hi-C and RNA-seq, it should be noted that the libraries are 2x150 bp reads.Also, the total amount of sequence data generated from these runs should be reported.The HiFi output was reported as "44-fold coverage" but I also think something more along the lines of total bases and number of reads generated should be included.
Minor comments/questions: "Inequivalve bivalve" -I think this term should be defined."The specimen was identified by Chris Fletcher" -Was there any morphological criteria or molecular analysis done to confirm the species ahead of sequencing?Is the Ultra II RNA Library Prep Kit a stranded library prep?Also, if it is not beyond the scope of detailing the genome, I think some detail about the quality of this data would be useful.Reviewer Expertise: Animal genomics, marine invertebrate evo-devo 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 Mimachlamys varia, xbMimVari1.1: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 975,381,475 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 (65,822,114 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (50,749,371 and 41,658,273 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/xbMimVari1.1/dataset/CANQLB01/snail.

Figure 3 .
Figure 3. Genome assembly of Mimachlamys varia, xbMimVari1.1: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/xbMimVari1.1/dataset/CANQLB01/blob.

Figure 5 .
Figure 5. Genome assembly of Mimachlamys varia, xbMimVari1.1:Hi-C contact map of the xbMimVari1.1 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=W3n8QQetR4WPxi2j_7zzcg.

Figure 4 .
Figure 4. Genome assembly of Mimachlamys varia, xbMimVari1.1: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/xbMimVari1.1/dataset/CANQLB01/ cumulative.
https://doi.org/10.21956/wellcomeopenres.21760.r63027© 2023 Martín-Durán 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.José María Martín-DuránSchool of Biological and Behavioural Sciences, Queen Mary University of London, London, UK This manuscript reports the chromosome-scale genome assembly of the bivalve Mimachlamys varia, a common member of the fauna on the shores of the UK, North Atlantic and Mediterranean seas.Produced with gold-standard methods of the DToL, this is a high-quality assembly that will be a valuable resource for many research fields, from evolutionary biology to ecology and marine biology.Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.

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:
Mainly, how much of it maps back to the genome assembly?It might be worth noting that 19 chromosomes are expected based on other chromosome-level assemblies of scallops and karyotypic analyses.Unless such comparison is beyond the scope of a Data Note in Wellcome Open Research.Did the MitoHiFi pipeline return a circular molecule, is that reported in the output?I know the rest of the annotation is yet to be done, but the mitochondrial genome assembly pipeline includes an automated annotation.Will this be the "official" annotation, if so, should those results be included here?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, however I have significant reservations, as outlined above.
Reviewer Report 24 July 2023