{"paper_id":"1638acf4-287b-45af-9f19-4e9fc528ff8b","body_text":"Data Paper (Biosciences)\nAuthor-formatted document posted on 26/03/2025\nPublished in a RIO article collection by decision of the collection editors.\nDOI: https://doi.org/10.3897/arphapreprints.e153920\nERGA-BGE genome of Acomys minous (Bate, 1906): the\nCrete spiny mouse, endemic to the island of Crete,\nGreece\n Petros Lymberakis,  Danae Karakasi, Manolis Papadimitrakis,  Rita Monteiro,  Astrid\nBöhne, Rosa Fernández, Nuria Escudero,  Jean-Marc Aury,  Alice Moussy, Corinne Cruaud,\nKarine Labadie, Sophie Mangenot, Caroline Belser, Lola Demirdjian,  Swati Sinha,  Leanne\nHaggerty, Fergal Martin, Patrick Wincker,  Pedro H. Oliveira,  Tom Brown\n\nERGA-BGE Genome Report - Acomys minous\n \n \n1 \nGENOME REPORT  \nERGA-BGE genome of Acomys minous  (Bate, 1906): \nthe Crete spiny mouse, endemic to the island of \nCrete, Greece \nPetros Lymberakis 1, Danae Karakasi 1,2, Manolis Papadimitrakis 1, Rita \nMonteiro3, Astrid Böhne 3, Rosa Fernández 4, Nuria Escudero4, Genoscope \nSequencing Technical Team 5, Alice Moussy 5, Corinne Cruaud 5, Karine \nLabadie5, Sophie Mangenot 5, Caroline Belser 6, Lola Demirdjian 6,  Swati \nSinha7, Leanne Haggerty 7, Fergal Martin 7, Patrick Wincker 6, Pedro H. \nOliveira6, Jean-Marc Aury6, Tom Brown8,9* \n1 Natural History Museum of Crete, School of Sciences and Engineering, University of Crete, Greece  \n2 Department of Biology, School of Sciences and Engineering, University of Crete, Vassilika Vouton, \nHeraklion, GR-70013, Greece \n3 Leibniz Institute for the Analysis of Biodiversity Change - Museum Koenig Bonn, Adenauerallee 127, \n53113 Bonn, Germany \n4 Metazoa Phylogenomics Lab, Institute for Evolutionary Biology (CSIC-UPF). Passeig marítim de la \nBarceloneta 37-49. 08003 Barcelona, Spain \n5 Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, Evry, 91057, \nFrance \n6 Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris -\nSaclay, 91057 Evry, France \n7 European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome \nCampus, Hinxton, Cambridge, UK \n8 Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Straße 17, 10315 Berlin, Germany \n9 Berlin Center for Genomics in Biodiversity Research (BeGenDiv), Koenigin-Luise-Str 6-8, 14195 \nBerlin, Germany \n* To whom correspondence should be addressed: brown@izw-berlin.de \nAbstract \nThe Acomys minous  reference genome offers a crucial resource for uncovering phylogenetic \nrelationships within the genus and its complex phylogeographic history . The entirety of the genome \nsequence was assembled into 20 contiguous chromosomal pseudomol ecules. This chromosome -\nlevel assembly encompasses 2.35 Gb, composed of 297 contigs and 113 scaffolds, with contig and \nscaffold N50 values of 29.3 Mb and 113 Mb, respectively. \nAuthor-formatted document posted on 26/03/2025. DOI:  https://doi.org/10.3897/arphapreprints.e153920\n\nERGA-BGE Genome Report - Acomys minous\n \n \n2 \nKeywords \nAcomys minous , genome assembly, European Reference Genome Atlas, Biodiv ersity Genomics \nEurope, Earth Biogenome Project, Muridae family, Crete spiny mouse, Agathopontikos \nIntroduction  \nAcomys minous is a species likely introduced to \nCrete  during the Middle Pleistocene (Barome et \nal., 2001). There has been a long controversy on its \ntaxonomic status, namely whether it should be \nconsidered as a distinct species or a subspecies (A. \ncahirinus minous ) of the North -African A. \ncahirinus (Aghová et al., 2019; Giagia -\nAthanasopoulou et al., 2011; Kryštufek et al., \n2009; Wilson et al., 2017). \nThe species has been recently assessed in Greece's \nregional Red Data list as Least Concern (Nikolaus, \n2024). \nBeing an introduced species notwithstanding,  A. \nminous has integrated into the envi ronment of \nCrete. It is part of the diet of many carnivores, \nmainly avian, and it consumes both plants and \ninvertebrate animals (Paragamian & Zivanovic, \n1989; Renaud et al., 2020; Wilson et al., 2017). \nDespite the importance of species from this  genus \nalongside its large distribution and abundance in \nlocal communities, the phylogeny and the species \nlimits in the genus are poorly resolved (Aghová et \nal., 2019). A high-quality reference genome for A. \nminous will enhance research for drawing a clearer \npicture on the phylogenetic and phylogeographic \nrelationships within the genus Acomys. \nThe generation of this reference resource was \ncoordinated by the European Reference Genome \nAtlas (ERGA) initiative’s Biodiversity Genomics \nEurope (BGE) project, supporting ERGA’s aims of \npromoting transnational cooperation to promote \nadvances in the application of genomics \ntechnologies to protect and restor e biodiversity \n(Mazzoni et al., 2023). \nMaterials & Methods \nERGA's sequencing strategy includes Oxford \nNanopore Technology (ONT) and/or Pacific \nBiosciences (PacBio) for long -read sequencing, \nalong with Hi -C sequencing for chromosomal \narchitecture, Illumina Paired -End (PE) for \npolishing (i.e. recommended for ONT -only \nassemblies), and RNA sequencing for \ntranscriptomic profiling, to facilitate genome \nassembly and annotation.  \nSample and Sampling Information  \nManolis Papadimitrakis sampled one specimen of \nfemale Acomys minous , identified by Petros \nLymberakis, determined based on expert \nassessment, from Almyros Gorge, Irakleio, Crete, \nGreece on 19 May 2019. Sampling was performed \nunder Presidential Decr ee 67/1981 issued by The \nGreek Government in Athens. Sampling was \nperformed using traps. The specimen was \neuthanized by increasing concentration of CO2 and \nsubsequently frozen at -80C. Until DNA \nextraction, samples were preserved at -80C.  \nVouchering information \nPhysical reference materials for the here sequenced \nspecimen have been deposited in Natural History \nMuseum of Crete, University of Crete \n(https://www.nhmc.uoc.gr/en/departments/vertebr\nates) under the accession number \nNHMC80.5.40.460.  \nFrozen reference tissue material of muscle is \navailable from the same individual at the Biobank \nNatural History Museum of Crete, University of \nCrete \n(https://www.nhmc.uoc.gr/en/departments/vertebr\nates) under the voucher ID NHMC.80.5.40.460. \nData Availability \nA. minous  and the related genomic study were \nassigned to Tree of Life ID (ToLID) ‘mAcoMin1’ \nAuthor-formatted document posted on 26/03/2025. DOI:  https://doi.org/10.3897/arphapreprints.e153920\n\nERGA-BGE Genome Report - Acomys minous\n \n \n3 \nand all sample, sequence, and assembly \ninformation are available under the umbrella \nBioProject PRJEB77214. The sample information \nis available at the following BioSample accessions: \nSAMEA112751364, SAMEA112751366, \nSAMEA112751371, SAMEA112751373 and \nSAMEA112751376. The genome assembly is \naccessible from  ENA under accession number \nGCA_964271855.1. Sequencing data produced as \npart of this project are available from ENA at the \nfollowing accessions: ERR12712697, \nERR13351238, ERR13351239 and \nERR13362573. Documentation related to the \ngenome assembly and curation can be found in the \nERGA Assembly Report (EAR) document \navailable at https://github.com/ERGA-\nconsortium/EARs/tree/main/Assembly_Reports/A\ncomys_minous/mAcoMin1. Further details and \ndata about the project are hosted on the ERGA \nportal at https://portal.erga-\nbiodiversity.eu/data_portal/Acomys%20minous. \nGenetic Information \nThe estimated genome size, based on ancestral \ntaxa, is 2.58 Gb. This is a diploid genome with a \nhaploid number of 20 chromosomes (2n=40) and \nXY sex chromosomes. All information for this \nspecies was retrieved from Genomes on a Tree \n(Challis et al., 2023). \nDNA/RNA processing \nDNA was extracted from 200 mg of muscle tissue \nusing a Genomic -tip 100/G kit (QIAGEN, MD, \nUSA) following manufacturer’s instructions. DNA \nfragment size selection was performed using Short \nRead Eliminator (PacBio, CA, USA). \nQuantification was performed using a Qubit \ndsDNA HS Assay kit (Thermo Fisher Scientific) \nand integrity was assessed in a FemtoPulse system \n(Agilent). DNA was stored at 4 °C until usage. \nRNA was extracted from muscle (50 mg) using the \nRNeasy Plus Universal kit (Qiagen) following \nmanufacturer instructions. Residual genomic DNA \nwas removed with 6U of TURBO DNase (2 U/μL) \n(Thermo Fisher Scientific). Quantification was \nperformed using a Qubit RNA HS Assay kit and \nintegrity was assessed in a Bioanalyzer system \n(Agilent). RNA was stored at -80 °C. \nLibrary Preparation and  Sequencing  \nLong-read DNA libraries were prepared with the \nSMRTbell prep kit 3.0 following manufacturers' \ninstructions and sequenced on a Revio system \n(PacBio). Hi -C libraries were generated from \nmuscle tissue using the Arima High Coverage HiC \nkit (following the Animal Tissues low input \nprotocol v01) and sequenced on a NovaSeq 6000 \ninstrument (Illumina) with 2x150 bp read length. \nPoly(A) RNA-Seq libraries were constructed using \nthe Illumina Stranded mRNA Prep, Ligation kit \n(Illumina) and sequenced on a  NovaSeq X+ \ninstrument (Illumina). In total 77.3 Gb PacBio \nHiFi, 211.3 Gb Illumina WGS, and 43.4 Gb HiC \ndata were sequenced to generate the assembly.  \nGenome Assembly Methods \nThe genome of Acomys minous  was assemb led \nusing the Genoscope GALOP pipeline \n(https://workflowhub.eu/workflows/1200). \nBriefly, raw PacBio HiFi reads were assembled \nusing Hifiasm v0.19.5 -r593. Retained haplotigs \nwere removed using purge_d ups v1.2.5 with \ndefault parameters and the proposed cutoffs. The \npurged assembly was scaffolded using YaHS v1.2 \nand assembled scaffolds were then curated through \nmanual inspection using PretextView v0.2.5 to \nremove false joins and incorporate sequences not  \nautomatically scaffolded into their respective \nlocations within the chromosomal \npseudomolecules. The mitochondrial genome was \nassembled as a single circular contig using Oatk \nv1.0 and included in the released assembly. \nSummary analysis of the released assembly was \nperformed using the ERGA -BGE Genome \nReport ASM Galaxy workflow \n(https://doi.org/10.48546/workflowhub.workflo\nw.1104.1). \nGenome Annotation Methods \nA gene set was generated using the Ensembl Gene \nAnnotation system (Aken et al., 2016) , primarily \nby aligning publicly available short-read RNA-seq \ndata from BioSample: SAMEA112751366 to the \nAuthor-formatted document posted on 26/03/2025. DOI:  https://doi.org/10.3897/arphapreprints.e153920\n\nERGA-BGE Genome Report - Acomys minous\n \n \n4 \ngenome. Gaps in the annotation we re filled via \nprotein-to-genome alignments of a select set of \nvertebrate proteins from UniProt (“UniProt: A \nWorldwide Hub of Protein Knowledge,” 2019) , \nwhich had experimental evidence at the protein or \ntranscript level. At each locus, data were \naggregated and consolidated, prioritising models \nderived from RNA-seq data, resulting in a final set \nof gene models and associated non -redundant \ntranscript sets. To distinguish true isoforms from \nfragments, the likelihood of each open reading \nframe (ORF) was evaluated against known \nvertebrate proteins. Low-quality transcript models, \nsuch as those showing evidence of fragmented \nORFs, were removed (thresholds needed). In cases \nwhere RNA-seq data were fragment ed or absent, \nhomology data were prioritised, favouring longer \ntranscripts with strong intron support from short -\nread data. The resulting gene models were \nclassified into three categories: protein -coding, \npseudogene, and long non -coding. Models with \nhits t o known proteins and few structural \nabnormalities were classified as protein -coding. \nModels with hits to known proteins but displaying \nabnormalities, such as the absence of a start codon, \nnon-canonical splicing, unusually small intron \nstructures (<75 bp), or excessive repeat coverage, \nwere reclassified as pseudogenes. Single -exon \nmodels with a corresponding multi -exon copy \nelsewhere in the genome were classified as \nprocessed (retrotransposed) pseudogenes. Models \nthat did not fit any of the previously descri bed \ncategories did not overlap protein -coding genes, \nand were constructed from transcriptomic data \nwere considered potential lncRNAs. Potential \nlncRNAs were further filtered to remove single -\nexon loci due to their unreliability. Putative \nmiRNAs were predicted by performing a BLAST \nsearch of miRBase (Kozomara et al., 2019) against \nthe genome, followed by RNAfold analysis \n(Gruber et al., 2008). Other small non-coding loci \nwere identified by scanning the genome with Rfam \n(Kalvari et al., 2018)  and passing the results \nthrough Infernal (Nawrocki & Eddy, 2013) . \nSummary analysis of the released annotation was \ncarried out using the ERGA-BGE Genome Report \nANNOT Galaxy workflow \n(https://doi.org/10.48546/workflowhub.workflow.\n1096.1). \nResults \nGenome Assembly \nThe genome assembly has a total length of \n2,350,237,540 bp in 113 scaffolds including the \nmitogenome (Figures 1 & 2), with a GC content of \n42.87%. The assembly has a contig N50 of \n29,281,624 bp and L50 of 23 and a scaffold N50 of \n122,851,966 bp and L50 of 9. The assembly has a \ntotal of 184 gaps, totaling 20.1 kb in cumulative \nsize. The single -copy gene content analysis using \nthe Glires database with BUSCO (Manni et al., \n2021) resulted in 95.3% completeness (94.0% \nsingle and 1.3% duplicated). 92.44% of reads k -\nmers were present in the assembly and the \nassembly has a base accuracy Quality Value (QV) \nof 59.0 as calculated by Merqury (Rhie et al., \n2020). \nGenome Annotation \nThe genome annotation consists of 19,865 protein-\ncoding genes with an associated 25,939 transcripts, \nin addition to 3,561 non -coding genes (Table 1). \nUsing the longest isoform per transcript, the single-\ncopy gene content analysis using the Glires \ndatabase with BUSCO resulted in 97.9% \ncompleteness. Using the OMAmer Myomorpha \ndatabase for OMArk (Nevers et al., 2024) resulted \nin 97.93% completeness a nd 97.8% consistency \n(Table 2). \n \n \n \n \n  \nAuthor-formatted document posted on 26/03/2025. DOI:  https://doi.org/10.3897/arphapreprints.e153920\n\nERGA-BGE Genome Report - Acomys minous\n \n \n5 \nTable 1. Statistics from assembled gene models \n No. genes No. \ntranscripts \nMean gene \nlength (bp) \nNo. single-\nexon genes \nMean exons \nper transcript \nmRNA 19,865 25,939 40,353 2,501 10.4 \npseudogene 416 416 11,537 18 9.7 \nsnoRNA 1,455 1,455 118 1,455 1 \nlncRNA 111 121 2,456 81 1.6 \nmiRNA 67 67 83 67 1 \nsnRNA 1,235 1,235 118 1,235 1 \nrRNA 118 118 354 118 1 \nscRNA 44 44 160 44 1 \nOther ncRNA 115 115 106-271 115 1 \nTable 2. Annotation completeness and consistency scores calculated by BUSCO run in protein \nmode (glires_odb10) and OMArk (Myomorpha) \n Complete Singular Duplicated Fragmented Missing \nBUSCO 13,514 (97.9%) 13,383 \n(97.0%) \n131 (0.9%) 44 (0.3%) 240 (1.8%) \nOMArk 15,571 \n(97.93%) \n15,243 \n(95.87%) \n328 (2.06%) - 329 (2.07%) \n Consistent Inconsistent Contaminants Unknown \nOMArk 19,653 \n(97.80%) \n345 (1.72%) 0 (0.0%) 97 (0.48%) \n \n \nAuthor-formatted document posted on 26/03/2025. DOI:  https://doi.org/10.3897/arphapreprints.e153920\n\nERGA-BGE Genome Report - Acomys minous\n \n \n6 \n \nFigure 1. Snail plot summary of assembly statistics. The main plot is divided into 1,000 size-ordered \nbins around the circumference, with each bin representing 0.1% of the 2,350,237,540 bp assembly. \nThe distribution of sequence lengths is shown in dark grey, with the plot radius scaled to the longest \nsequence present in the assembly (187,704,340 bp, shown in red). Orange and pale -orange arcs \nshow the scaffold N50 and N90 sequence lengths (122,851,966 and 71,069,054 bp), respectively. \nThe pale grey spiral shows the cu mulative sequence count on a log -scale, with white scale lines \nshowing successive orders of magnitude. The blue and pale-blue area around the outside of the plot \nshows the distribution of GC, AT, and N percentages in the same bins as the inner plot. A summary \nof complete, fragmented, duplicated, and missing BUSCO genes found in the assembled genome \nfrom the Glires database (odb10) is shown in the top right. \nAuthor-formatted document posted on 26/03/2025. DOI:  https://doi.org/10.3897/arphapreprints.e153920\n\nERGA-BGE Genome Report - Acomys minous\n \n \n7 \n \nFigure 2.  Hi-C contact map showing spatial interactions between regions of the genome. The \ndiagonal corresponds to intra -chromosomal contacts, depicting chromosome boundaries. The \nfrequency of contacts is shown on a logarithmic heatmap scale. Hi-C matrix bins were merged into \na 25 kb bin size for plotting. \n \n \n \n \n \nAuthor-formatted document posted on 26/03/2025. DOI:  https://doi.org/10.3897/arphapreprints.e153920\n\nERGA-BGE Genome Report - Acomys minous\n \n \n8 \nAcknowledgements \nWe would like to acknowledge the assembly reviewer, Tyler Alioto, from the Centro Nacional de Análisis \nGenómico (CNAG). The authors acknowledge the support of the Freiburg Galaxy Team: Saim Momin and \nBjörn Grüning, Bioinformatics, University of Freiburg (Germany), funded by the German Federal Ministry \nof Education and Research BMBF grant 031 A538A de.NBI-RBC and the Ministry of Science, Research and \nthe Arts Baden-Württemberg (MWK) within the framework of LIBIS/de.NBI Freiburg. \nConflict of Interest \nThe authors declare no conflict of interest related to this study. The funding sources had no involvement in \nthe study design, collection, analysis, or interpretation of data; in the writing of the manuscript; or in the \ndecision to submit the article for publication. All authors have part icipated sufficiently in the work to take \npublic responsibility for the content and agree to the submission of this manuscript. \nFunder Information \nThis project received funding from Horizon Europe under the Biodiversity, Circular Economy and \nEnvironment (REA.B.3); co-funded by the Swiss State Secretariat for Education, Research and Innovation \n(SERI) under contract numbers 22.00173 and 24.00054; and by the UK Research and Innovation (UKRI) \nunder the Department for Business, Energy and Industrial Strategy’s H orizon Europe Guarantee Scheme.  \nThis work was supported by the Genoscope, the Commissariat à l'Énergie Atomique et aux Énergies \nAlternatives (CEA), France Génomique (ANR -10-INBS-09-08), and the exploratory research programme \n‘ATLASea: Atlas of marine genomes’ and its targeted project SEQ-Sea (ANR-22-EXAT-0003-SEQ-Sea).. \nAuthor Contributions \nMP and PL collected the species, PL identified the species, DK and MP sampled and preserved biological \nmaterial and provided metadata, RM and AS provided sampling and metadata support and management, the \nGenomescope Sequencing Team extracted DNA, prepared libraries, and performed sequencing under the \nsupervision of AM, CC, KL, PHO and PW, LD SM, CB and JMA performed genome assembly and curation, \nTB generated the analysis  and report. All authors contributed to the writing, review, and editing of this \ngenome note and read and approved the final version. This work is part of the species assigned to Genoscope, \nwhich was instrumental in the wet lab, sequencing, and assembly processes, and represents a key contribution \nto BGE's outputs \nAuthor Information \nMembers of the Genoscope Sequencing Technical Team are listed here: \nhttps://doi.org/10.5281/zenodo.14611490\n \n  \nAuthor-formatted document posted on 26/03/2025. DOI:  https://doi.org/10.3897/arphapreprints.e153920\n\nERGA-BGE Genome Report - Acomys minous\n \n \n9 \nLiterature Cited\n \nAghová, T., Palupčíková, K., Šumbera, R., Frynta, D., Lavrenchenko, L. A., Meheretu, Y., Sádlová, J., \nVotýpka, J., Mbau, J. S., Modrý, D., & Bryja, J. (2019). 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Handbook of the mammals of the world. \nLynx Edicions : Conservation International : IUCN. \nAuthor-formatted document posted on 26/03/2025. DOI:  https://doi.org/10.3897/arphapreprints.e153920\n\nAuthor-formatted document posted on 26/03/2025. DOI:  https://doi.org/10.3897/arphapreprints.e153920\n\nAuthor-formatted document posted on 26/03/2025. DOI:  https://doi.org/10.3897/arphapreprints.e153920","source_license":"CC-BY-4.0","license_restricted":false}