The genome sequence of the northern bat, Eptesicus nilssonii (Keyserling & Blasius, 1839)

We present a genome assembly from an individual Eptesicus nilssonii (the northern bat; Chordata; Mammalia; Chiroptera; Vespertilionidae), derived from the placental tissue of a pregnancy that resulted a male pup. The genome sequence is 2,064.1 megabases in span. Most of the assembly is scaffolded into 26 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 17.04 kilobases in length.


Background
The northern bat, Eptesicus nilssonii, is a medium-sized, northern Palearctic bat species with a distribution ranging from Scandinavia and the Alps in the west to Kamchatka and Japan in the east.In Europe it is widely distributed in the north and east, but in central Europe it is restricted to forested areas at higher elevations (Gerell & Rydell, 2001;López-Baucells & Burgin, 2019;Rydell, 1993;Suominen et al., 2022).It has the most northern distribution of any bat species in the world, with a breeding population as far north as 69°N (Rydell et al., 1994;Speakman et al., 2000).It is assessed as Least Concern for the global IUCN Red list (Coroiu, 2016), but winter and summer census data from both Sweden and Norway indicate a recent population decline (Ahlén & Ahlén, 2015;Eldegard et al., 2021;Frafjord, 2013;Rydell et al., 2019;Rydell et al., 2020), and therefore the species is listed as Near Threatened in Sweden (De Jong et al., 2020) and Vulnerable in Norway (Eldegard et al., 2021).Climate change and the disuse of the insect-attracting mercury-vapour streetlights most likely play a crucial role in the decline (Eldegard et al., 2021).
Northern bats are adapted to short, light, cool summer nights and long, cold winters: they are relatively light tolerant (Rydell, 1993).Juvenile bats undertake their first outdoor flights only two weeks after birth (Rydell, 1992;Rydell, 1993), and animals of both sexes make extensive use of torpor in the active season (Fjelldal et al., 2023;Rydell, 1993;Siivonen & Wermundsen, 2008).During winter they express longer average bouts of torpor than other species (Solomonov et al., 2010), are found just above or sometimes even below freezing temperatures (Masing & Lutsar, 2007;Siivonen & Wermundsen, 2008;Wermundsen & Siivonen, 2010) and have, inter alia, a relatively high peripheral lymphocyte count and a high vitamin E content in the liver (Ilyukha et al., 2015).
In Western Europe, nursery colonies are mainly found in buildings (Rydell, 1993).They regularly hibernate in human-made underground sites, but recent studies have made it plausible that the majority use natural structures like screes, glacial erratics and bedrock crevices (Blomberg et al., 2021;Frafjord, 2007;Michaelsen et al., 2013).
We present a chromosomally complete genome sequence for Eptesicus nilssonii.The sequence is based on a male placenta, retrieved shortly after birth from a monitored roost in Norway (Slattum, Nittedal municipality, Akershus county.This sampling is part of the Bat1K Project (Teeling et al., 2018) and the Darwin Tree of Life Project (DToL).The Bat1K is a collaborative effort to sequence all extant bat species, and DToL aims to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.
The use of the placenta in this context, where neither mother nor pup are euthanised, offers a more animal-friendly alternative for obtaining samples for genome analyses.Additionally, we hope that this genome may assist in uncovering the genetic basis for environmental adaptations to live in a cold climate.

Genome sequence report
The genome was sequenced from Eptesicus nilssonii placental tissue collected from a monitored roost in Akershus, Norway 67 (60.02, 10.9).A total of 33-fold coverage in Pacific Biosciences single-molecule HiFi long was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 49 missing joins or misjoins and removed 18 haplotypic duplications, reducing the assembly length by 0.76% and the scaffold number by 16.87%.
The final assembly has a total length of 2,064.1 Mb in 206 sequence scaffolds with a scaffold N50 of 98.0 Mb (Table 1).Most (98.85%) of the assembly sequence was assigned to 26 chromosomal-level scaffolds, representing 24 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.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/59451.
The specimen was taken from a maternity colony by removing the placenta from the uterus about one hour after the birth of the pup.It was placed immediately in an Eppendorf tube, and the tube was placed in dry ice in a -20°C freezer and transferred to a -80°C freezer within 24 h.The specimen was collected and identified by Jeroen van der Kooij (independent researcher).
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The mEptNil1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.The placenta 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 placental tissue of mEptNil1 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 at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from placental tissue of mEptNil1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

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 (Zhou et al., 2023).The assembly was checked for contamination and corrected as described previously (Howe et al., 2021).Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.Table 3. Software tools: versions and sources.
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 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 material

Wenhua Yu
Guangzhou University, Guangzhou, China Xiaoyun Wang School of Life Sciences, Guangzhou University (Ringgold ID: 47875), Guangzhou, Guangdong, China The authors have successfully obtained the genome of the Eptesicus nilssonii using the placental tissue.Given the current relative scarcity of complete genomic data within the Chiroptera, this study undoubtedly provides meaningful genomic information for subsequent research.
The manuscript is well-written, and the analytical methods used are all reasonable and high standard, serving as an ideal framework for future genomic report-like research outcomes.At the current stage, I only recommend that the authors further describe the introduction regarding the species' adaptive evolution to low temperatures at the genomic level, which would also lay the foundation for future research and reporting.
minor issue: In Table 2, an error in the sequencing 7 and 8.

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 This manuscript reports the first genome sequence of the northern bat, Eptersicus nilssonii, which is useful for studying the environmental adaptations of this species.In particular, this study used placental tissue for genome sequencing, providing an animal-friendly alternative for obtaining samples for genome analyses.The tables and figures are clear, and the manuscript is easy to read.However, I still have some minor comments for the manuscript.
(1) In the manuscript, when Eptesicus nilssonii first appears, write the full name, and for subsequent mentions, provide the abbreviation, E. nilssonii.
(2) For the first sentence "The genome was sequenced from Eptesicus nilssonii placental tissue collected from a monitored roost in Akershus, Norway 67 (60.02, 10.9)." in "Genome sequence report", I think you can remove it to the fourth paragraph and integrate with the second sentence.Please indicate N and E.
(3) For Figures 3 and 4, I think you can rotate the description of the y-axis counterclockwise by 180 degrees.

Xiuguang Mao
East China Normal University, Shanghai, China van der Kooij et al. present a high-quality chromosome-scale assembly for a male Eptesicus nilssonii .The processes of genome sequencing, assembly and assessment follow a high standard pipeline.The total number of chromosomal-level scaffolds corresponds to the haploid chromosome number of this species (2n=50).This reference genome, together with another one from a female individual (GenBank accession: GCA_030846915.1), will provide valuable resources to study the genetic basis of cold adaptation.Three minor comments: Can authors say a little bit about how they assign the sex chromosomes (X and Y chromosome)? 1.
Can authors provide specific amounts of sequencing data for HiFi data (? Gb) and Hi-C data (? Gb) used in genome assembly? 2.
In Column "Chromosome" in Table 2, "8" and "7" should be reordered if chromosomes are named in the order of size.

3.
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.

Figure 1 .
Figure 1.Photographs of Eptesicus nilssonii mother, Nikki, and her pup, Karl, captured by Jeroen van der Kooij.The placenta from Karl's birth was sequenced for this study.A. Karl at 2 days, B. Nikki with Karl at 6 days old.C. Nikki with Karl at 12 days old, D. Karl, 12 days old.

Figure 2 .
Figure 2. Genome assembly of Eptesicus nilssonii, mEptNil1.1: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 2,064,119,045 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 (133,114,493 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (98,018,206 and 52,996,688 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 laurasiatheria_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/mEptNil1.1/dataset/CATOCV01/snail.

Figure 3 .
Figure 3. Genome assembly of Eptesicus nilssonii, mEptNil1.1: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/mEptNil1.1/dataset/CATOCV01/blob.

Figure 4 .
Figure 4. Genome assembly of Eptesicus nilssonii, mEptNil1.1: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/mEptNil1.1/dataset/CATOCV01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Eptesicus nilssonii, mEptNil1.1:Hi-C contact map of the mEptNil1.1 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=X_AxuaqeQXqkZTMYsTSOwg.

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.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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.