The genome sequence of the wood mouse, Apodemus sylvaticus (Linnaeus, 1758)

We present a genome assembly from an individual male Apodemus sylvaticus (the wood mouse; Chordata; Mammalia; Rodentia; Muridae). The genome sequence is 2,889.8 megabases in span. Most of the assembly is scaffolded into 25 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 16.31 kilobases in length.


Background
The wood mouse (or long-tailed field mouse), Apodemus sylvaticus (Figure 1), is an extremely common and widespread species of mouse with a broad geographic range spanning most of Europe as well as parts of northern Africa, from sea level to nearly 2 km in altitude (IUCN, 2022).Wood mice inhabit a wide variety of habitats, including woodlands, grassland, hedgerows, urban environments and gardens, arable land, moorland and sand dunes.Throughout much of their range, wood mice are sympatric with the closely related species Apodemus flavicollis (yellow-necked mouse) and can be distinguished from this species by the lack of a continuous band of brown fur across the neck and typically smaller adult size.Wood mice are flexible omnivores, though are usually primarily granivorous, eating seeds from trees including oak, beech, ash, hawthorn and sycamore, but also invertebrates, fruit and fungi depending on availability (Khammes & Aulagnier, 2007;Watts, 1968;Zubaid & Gorman, 1991).
Wood mice build extensive underground burrows in which they sleep and cache food (Jennings, 1975).They are almost entirely nocturnal, emerging to make foraging trips throughout the night (Wolton, 1985).They have small, overlapping home ranges that vary in size and overlap with sex and the stage of the breeding season (Godsall et al., 2014;Wolton, 1985) Their mating system is promiscuous, with males typically mating multiple females and litters often containing pups from multiple males (Booth et al., 2007).
Wood mice have become a key model species in ecology and evolutionary biology due to their abundance, ease of capture, and ability to be maintained in captivity.For example, they are frequently used in studies of parasite and microbiome ecology (Knowles et al., 2013;Marsh et al., 2022;Raulo et al., 2021;Sweeny et al., 2021), eco-immunology (Babayan et al., 2018;Clerc et al., 2019), sexual selection (Moore et al., 2002) and behavioural ecology (Stopka & Macdonald, 2003).They are also of interest as relatively anthropophilic reservoirs of zoonotic pathogens (Borg et al., 2017;Crouch et al., 1995).This species commonly undergoes dramatic seasonal bottlenecks, whereby over 80% of the population can die between breeding seasons (Flowerdew, 1985).Despite these frequent bottlenecks, it has been shown that wood mouse populations can be relatively stable in genetic composition (Booth et al., 2005).
The genome of the wood mouse, Apodemus sylvaticus, was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.Here we present a chromosomally complete genome sequence for Apodemus sylvaticus, based on ear clipping samples from one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Apodemus sylvaticus (Figure 1) caught in Wytham Woods, Oxfordshire, UK (51.77,.A total of 25-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 16-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 138 missing joins or misjoins and removed 5 haplotypic duplications, reducing the scaffold number by 11.39%. The final assembly has a total length of 2889.8Mb in 497 sequence scaffolds with a scaffold N50 of 120.9 Mb (Table 1).Most (95.59%) of the assembly sequence was assigned to 25 chromosomal-level scaffolds, representing 23 autosomes and the X and Y sex chromosomes.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.

Sample acquisition and nucleic acid extraction
Two Apodemus sylvaticus, a male and a female, were livecaught in baited Sherman traps in Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2020-10-28.The animals were collected  and identified by Aura Raulo and Sarah Knowles (University of Oxford).A small ear clip was taken from each mouse using a sterilised ear punch, and snap-frozen on dry ice (under Home Office license PPL PB0178858).The specimen used for DNA sequencing was the male Apodemus sylvaticus (specimen ID Ox000983, ToLID mApoSyl1), while the female specimen ID Ox000985, ToLID mApoSyl2) was used for Hi-C data.Both animals were released at their site of capture within several hours of trap collection.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The mApoSyl1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.The tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using the 0.8X AMpure XP purification 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 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

Software tool Version
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 The article presents the genome assembly and annotation of Apodemus sylvaticus, commonly known as the wood mouse.The study is part of the Darwin Tree of Life Project, aiming to sequence the genomes of all eukaryotic species in the British Isles.The research involved capturing two specimens, extracting high molecular weight DNA, sequencing, and performing various bioinformatics analyses to produce a high-quality genome assembly.The assembly provides significant insights into the genetic composition and structure of the species, offering valuable data for comparative genomics and evolutionary studies.
The article clearly outlines the rationale behind creating the dataset, emphasizing the importance of sequencing Apodemus sylvaticus as part of the broader goal to understand the biodiversity of the British Isles and contribute to the Darwin Tree of Life Project.The authors also highlight the species' ecological significance and the potential insights the genomic data can provide into evolutionary biology and conservation.
The protocols employed in the study are standard and appropriate for genomic sequencing and assembly.The authors utilized high-quality DNA extraction methods, various sequencing technologies, and advanced bioinformatics tools for assembly and annotation.The detailed description of the procedures, including the use of tools like BlobToolKit and BUSCO, ensures that the work is technically robust.
The article provides comprehensive details of the methods and materials used, including specifics about DNA extraction, sequencing, and data analysis.The inclusion of software versions and sources further aids in replicability.The study's methodology is meticulously documented, making it possible for other researchers to replicate the work under similar conditions.
The datasets are presented clearly and are made accessible through various online platforms, including BlobToolKit and the Darwin Tree of Life Project's repositories.Interactive visualizations and comprehensive metadata ensure that the data is usable for further research and analyses.
While the rationale is clear, a more in-depth discussion on the specific ecological and evolutionary questions the dataset can address would enhance the relevance and context.
The article successfully meets the standards of scientific reporting, with clear rationale, appropriate protocols, comprehensive methodological details, and accessible data presentation.
The study is scientifically sound and provides a valuable resource for the scientific community.The minor improvements suggested are intended to further strengthen the clarity and utility of the work.
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: conservation genetics, genomics, conservation biology, ecology 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.
generating a new genome assembly.The sequencing was performed using PacBio HiFi, 10x Genomics, and Hi-C data.The resulting assembly meets and exceeds general benchmarks for high-quality assemblies, including scores for BUSCO, k-mer completeness, and the percentage of the assembly mapped to chromosomes.All raw and analysed datasets are deposited in publicly available repositories.
I see no need for additional improvements or revisions to the manuscript.

Xiuguang Mao
East China Normal University, Shanghai, China This manuscript presents a high-quality chromosome-scale assembly for one male Apodemus sylvaticus, which will be very useful for studies in ecology and evolutionary biology.I have two minor comment: "Most of the assembly sequence was assigned to 25 chromosomal-level scaffolds, representing 23 autosomes and the X and Y sex chromosomes."In Methods the authors should provide details about how they determine the sex chromosomes, such as by performing genome alignments with previous high-quality genomes or comparing the difference of sequencing coverage between autosomes and sex chromosomes?
In Table 2 chromosome 16 is longer than chromosome 15 in length.Thus, to make sure that "chromosomes are shown in order of size", Chr15 and Chr16 should be reordered.

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: Comparative genomics, genome assembly, evolutionary biology, speciation, bats 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.

Daniel Garcia-Souto
University of Santiago de Compostela, Santiago de Compostela, Galicia, Spain The presented genome assembly of the wood mouse, Apodemus sylvaticus, showcases an exceptional quality both in continuity and annotation.The assembly spans into 25 scaffolded chromosomal pseudomolecules, demonstrating a high level of continuity.Furthermore, the inclusion of both X and Y sex chromosomes and the mitochondrial genome, reflects the most complete version of the genome of this species.
This genome assembly is of particular significance due to the known presence of accessory chromosomes and Robertsonian translocations within populations of this organism, which make Apodemus sylvaticus an interesting subject for genetic and evolutionary studies.The high-quality assembly provided here will facilitate further research into these processes, enhancing our understanding of genome architecture and evolution in rodent species.
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: Cytogenetics, Genomics, Evolution, Karyotyping 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 1 .
Figure 1. A. Photograph of a juvenile Apodemus sylvaticus (by BlueBreezeWiki) (not the specimen used for genome sequencing).B. Ear clippings of individual mApoSyl1 used for genome sequencing and Hi-C data.

Figure 2 .
Figure 2. Genome assembly of Apodemus sylvaticus, mApoSyl1.1: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 2,889,801,511 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 (211,764,336 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (120,938,251 and 63,776,737 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 glires_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/mApoSyl1.1/dataset/CAMXCG01/snail.

Figure 3 .
Figure 3. Genome assembly of Apodemus sylvaticus, mApoSyl1.1: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/mApoSyl1.1/dataset/CAMXCG01/blob.

Figure 4 .
Figure 4. Genome assembly of Apodemus sylvaticus, mApoSyl1.1: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/mApoSyl1.1/dataset/CAMXCG01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Apodemus sylvaticus, mApoSyl1.1:Hi-C contact map of the mApoSyl1.1 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=N_6vvYjRR-SABvzgRaoJkQ.

Table 3
contains a list of relevant software tool versions and sources.

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: Population genomics, Conservation, 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.
Reviewer Report 01 August 2024 https://doi.org/10.21956/wellcomeopenres.22147.r89636© 2024 Mao X.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.