The genome sequence of the Soprano Pipistrelle, Pipistrellus pygmaeus (Leach, 1825)

We present a genome assembly from an individual male Pipistrellus pygmaeus (the Soprano Pipistrelle; Chordata; Mammalia; Chiroptera; Vespertilionidae). The genome sequence is 1,895.1 megabases in span. Most of the assembly is scaffolded into 23 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 17.18 kilobases in length.


Background
The Soprano pipistrelle (Pipistrellus pygmaeus) (Figure 1) is a common bat found throughout most of Europe where it lives in urbanised, as well as in a variety of riparian habitats, often near lakes or large rivers (Davidson-Watts et al., 2006).With an average adult weight of 3-4 g, it is the smallest European species of bat.During the summer, breeding females may aggregate into large colonies of up to a thousand individuals (Oakeley & Jones, 1998).Nursery colonies are typically installed under roofs, attics, behind shutters or in any kind of cracks in buildings.Because of this anthropophilic behaviour, the Soprano Pipistrelle is often abundant and does not suffer from any major threats in Europe.It is classified as Least Concern (LC) under IUCN criteria.
Populations in parts of their range appear to be migratory (e.g. in the Balkans) whereas others, such as in Western Europe, do not seem to engage in significant migrations (Bryja et al., 2008).Interestingly, this bat has been known to science only very recently (its species status was recognised in 1997 (Barratt et al., 1997)) owing to its species typical echolocation calls, which are produced at 55 kHz and distinguish it from those of the Common Pipistrelle (Pipistrellus pipistrellus), calling at 45 kHz (Barlow & Jones, 1997).Morphologically, however, both species are extremely similar, and may live in sympatry in many parts of Europe (Davidson-Watts et al., 2006).
The genome of this species has not been characterised previously, but extensive phylogeographic studies based on mitochondrial (Hulva et al., 2004) or few nuclear loci (Bryja et al., 2008) have been published in order to understand its molecular distinction from the sibling species P. pipistrellus.
This new, complete genome of a P. pygmaeus will therefore provide an unprecedented resolution into the evolution of these two cryptic species, given that a comparative genome for the common pipistrelle is already available.
We present a chromosomally complete genome sequence for Pipistrellus pygmaeus, based on one male specimen from Geneva, Switzerland, as part of the Darwin Tree of Life Project and Bat1K Project (Teeling et al., 2018).This project is a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.

Genome sequence report
The genome was sequenced from one male Pipistrellus pygmaeus collected from Geneva, Switzerland (46.19,6.17).A total of 36-fold coverage in Pacific Biosciences singlemolecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 16 missing joins or misjoins and removed 7 haplotypic duplications, reducing the assembly length by 0.43% and the scaffold number by 6.18%, and increasing the scaffold N50 by 0.5%.
The final assembly has a total length of 1,895.1 Mb in 242 sequence scaffolds with a scaffold N50 of 89.5 Mb (Table 1).Most (97.34%) of the assembly sequence was assigned to 23 chromosomal-level scaffolds, representing 21 autosomes and the X and Y sex chromosomes.The sex chromosomes were assigned by coverage statistics.Chromosomescale 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/246814.

Sample acquisition and nucleic acid extraction
The specimen used for DNA sequencing was a male P. pygmaeus (specimen ID SAN0001697, ToLID mPipPyg2), which was collected in Geneva, Switzerland (latitude 46.19, longitude 6.17) on 2021-06-17.The specimen used for RNA sequencing was a female P. pygmaeus (ToLID mPipPyg3), collected in Geneva, Switzerland (latitude 46.19, 6.12) on 2016-10-03.Both specimens were injured, euthanised bats, from an urban city habitat.The specimens were collected and identified by Manuel Ruedi (Natural History Museum     Fragment size distribution was evaluated by running the sample on the FemtoPulse system. RNA was extracted from liver tissue of mPipPyg3 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.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

Software tool Version
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 Overall the study serves as an important guide for researchers and students who are interested in high quality genome sequencing and assembly of natural populations.
Minor comments: I could not find the method for generating 10X genomics read data in the sequencing section.It will be good if the authors can add a short description of the method.
For genome assembly, curation and evaluation I would request the authors to add maybe in brief, some of the important parameters that were used for each program.Also I will request the authors to publicly share the scripts ( maybe though github page) necessary to perform the analyses.

Matthew Christmas
Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Uppsala County, Sweden Ruedi et al. provide a description of the chromosome-level assembly of the Pipstrellus pygmaeus genome, including the assembly of PacBio HiFi reads, assembly polishing, scaffolding, and mitogenome assembly.The assembly pipeline is appropriate and all assembly steps are clearly described.This genome provides an excellent resource as a basis for studying the evolution of this species and its divergence from other chiropterans, including the closely related Pipistrellus pipistrellus.
One minor comment: "The estimated Quality Value (QV) of the final assembly is 61" -it would be good to provide context for this, does a score of 61 suggest high quality?
○ 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: Evolutionary genomics, population genomics, comparative genomics, genome assembly 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 Pipistrellus pygmaeus, mPipPyg2.1: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 1,895,125,685 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 (212,679,785 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (89,507,134 and 49,778,105 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/mPipPyg2.1/dataset/CATLKC01/snail.

Figure 3 .
Figure 3. Genome assembly of Pipistrellus pygmaeus, mPipPyg2.1: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/mPipPyg2.1/dataset/CATLKC01/blob.

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 muscle tissue of mPipPyg2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Figure 4 .
Figure 4. Genome assembly of Pipistrellus pygmaeus, mPipPyg2.1: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/mPipPyg2.1/dataset/CATLKC01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Pipistrellus pygmaeus, mPipPyg2.1:Hi-C contact map of the mPipPyg2.1 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=CzJBxflARPCHULHU6AtV-A.

Open Peer Review Current Peer Review Status: Version 1
The genome sequence is released openly for reuse.The Pipistrellus pygmaeus genome sequencing initiative is part of the Darwin Tree of Life (DToL) project.All raw sequence data and the assembly have been deposited in INSDC databases.The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute.Raw data and assembly accession identifiers are reported in Table1.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.Reudi et al. has presented a high quality assembly the Soprano Pipistrelle, Pipistrellus pygmaeus.The authors have assembled the genome into mitochondrial genome and chromosomal pseudomolecules including sex chromosomes.

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.
https://doi.org/10.21956/wellcomeopenres.22030.r81217©2024Mao X.Xiuguang MaoEast China Normal University, Shanghai, China Ruedi et al. present a high-quality chromosome-scale assembly for Pipistrellus pygmaeus.The ends of most of the chromosomal-scale scaffolds have telomere sequences, supporting the completeness of this assembly.In addition, the total number of chromosomal-level scaffolds corresponds to the haploid chromosome number of this species (2n=44).This genome provides valuable resource to study the genomic divergence of this species from other Pipistrellus (P.pipistrellus, P. kuhlii and P. nathusii) which also have high-quality assemblies.

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.