The genome sequence of the White-legged damselfly, Platycnemis pennipes (Pallas, 1771)

We present a genome assembly from an individual male Platycnemis pennipes (the White-legged damselfly; Arthropoda; Insecta; Odonata; Platycnemididae). The genome sequence is 1793.3 megabases in span. Most of the assembly is scaffolded into 13 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 15.42 kilobases in length.


Background
The White-legged damselfly, Platycnemis pennipes, occurs throughout most of western and central Europe, with a southern distribution in England and Wales.It is the only species in the family Platycnemididae resident in the UK.It is found mainly along slow-flowing lowland streams and rivers, though it can sometimes be found on canals or ponds (Brooks & Cham, 2014).The species is vulnerable to pollution, and especially vulnerable to physical disturbance of its bankside habitats (Corbet & Brooks, 2008).Unusual among non-demoiselle damselflies, males of the white-legged damselfly use their white legs in courtship displays for females and threat displays to other males (Brooks & Cham, 2014).Once mated, tandem pairs will often congregate for oviposition (Martens, 1996).Adults are on the wing from late May to mid-August.
The genome of P. pennipes 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 Platycnemis pennipes, based on one male specimen from Hever Castle, England.

Genome sequence report
The genome was sequenced from one male Platycnemis pennipes (Figure 1) collected from Hever Castle,England (51.19,0.12).A total of 29-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 37 missing joins or mis-joins and removed 12 haplotypic duplications, reducing the assembly length by 0.58% and the scaffold number by 5.43%.
The final assembly has a total length of 1793.3Mb in 87 sequence scaffolds with a scaffold N50 of 144.8 Mb (Table 1).Most (99.84%) of the assembly sequence was assigned to 13 chromosomal-level scaffolds, representing 12 autosomes and the X sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).The specimen was assigned as an XO male, since there was half-coverage of the X chromosome on the Hi-C map, with Hi-C data from a female specimen.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.The estimated Quality Value (QV) of the final assembly is 66.4 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 completeness of 97.1% (single = 96.4%,duplicated = 0.7%), using the insecta_odb10 reference set (n = 1,367).
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/126231.

Sample acquisition and nucleic acid extraction
The data presented here were derived from three Platycnemis pennipes specimens collected from the estate grounds at Hever Castle, England (latitude 51.19, longitude 0.12) on 2020-08-27 using an aerial net.The specimens were collected by Benjamin Price and Louise Allan (Natural History Museum) and identified by Benjamin Price.The specimens were preserved in liquid nitrogen.The specimen used for DNA sequencing was a male (specimen ID NHMUK014444682, ToLID ioPlaPenn1), a female specimen (specimen ID NHMUK014444686, ToLID ioPlaPenn2) was used for Hi-C scaffolding, and another male specimen (specimen ID NHMUK014444687, ToLID ioPlaPenn3) was used for RNA sequencing.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ioPlaPenn1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Thorax 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.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 thorax tissue of ioPlaPenn3 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 head tissue of ioPlaPenn2 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 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 done using Nextflow (Di

Rhys Parry
The University of Queensland, Saint Lucia, Queensland, Australia Price and Allan present the genome sequence of the White-legged damselfly, Platycnemis pennipes, assembled with two samples (one male, one female) and one male specimen for RNA sequencing.I have slight reservations about positive identification of the samples (male and female) and confidence of identification either through morphology or independent assembly of the mtDNA from both samples and then comparisons but it is probably unnecessary.The assembly appears to be of high quality with good BUSCO scores, the data is available, and the pipeline for assembly is sound.

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Metagenomics and genome assemblies of bacteria/viruses from environmental sources.
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.

Jeffrey Marcus
University of Manitoba, Winnipeg, Manitoba, Canada In this manuscript, the authors describe the sequencing and assembly of the Platycnemis pennipes genome using nucleic acids from 3 specimens collected in the UK.The primary genome sequence assembly is from a single male specimen, from which proposed chromosomal pseudomolecule sequences were proposed for 12 autosomes, a sex chromosome, and a complete mitochondrial genome.A female specimen had DNA sequenced to assist in identifying the sex chromosomes in the assembly and to infer the sex determining mechanism in this species.Males appear to be XO and females XX in P. pennipes.An additional male specimen was used for RNA sequencing, presumably to assist in identifying coding sequences in the assembled genome.Overall, this is a solid contribution to the scientific literature.Some suggestions to the authors: Background: "The White-legged damselfly, Platycnemis pennipes, occurs throughout most of western and central Europe, with a southern distribution in England and Wales."It is not clear from the text whether the authors are suggesting that England and Wales are the southern parts of the European distribution of this species or instead that they are suggesting that this species can be found in the southern parts of England and Wales in addition to portions of continental Europe. 1.
Genome sequence report: "The genome was sequenced from one male Platycnemis pennipes…".The ID and ToLID numbers for the relevant specimen should be included at first mention.

2.
Genome sequence report: "The specimen was assigned as an XO male, since there was halfcoverage of the X chromosome on the Hi-C map with Hi-C data from a female specimen."This is the first mention of the existence of a female specimen, let alone the sequencing of that specimen, in the document.Consequently, this statement is confusing and is not clarified until 3 paragraphs later when this female specimen is described in additional detail in the Methods.

3.
Figure 5. Numbering the chromosomes in this figure would be helpful to those trying to decipher it, either along the axes, or adjacent to the dark squares for each chromosome rendered by HiGlass.

4.
I agree with Xiaohan Shu that additional description of the annotation process for the nuclear and mitochondrial genomes would be helpful to readers.As it currently stands, the methods for annotation are clear, but not the results.

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?Partly Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Evolutionary biology of insects, phylogenomics 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, however I have significant reservations, as outlined above.
Reviewer Report 26 February 2024 https://doi.org/10.21956/wellcomeopenres.21899.r74488 © 2024 Shu 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.

Xiaohan Shu
Zhejiang University, Hangzhou, Zhejiang, China This manuscript reports the chromosomal-level genome of the White-legged damselfly, Platycnemis pennipes, providing a new genomic resource for damselfly research.The authors also report the mitochondrial genome of P. pennipes.It is a good work.
Below are suggestions for modifications: 1.The authors could provide annotation information for the assembled genome and the mitochondrial genome.2. In Figure 5, it would be beneficial to label each chromosome with its number.Additionally, placing the X chromosome at the end may be more logical.

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: Insect genomics, Ecology & Evolution 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 Platycnemis pennipes, ioPlaPenn1.1: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 1,793,356,121 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 (174,044,382 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (144,837,943 and 108,313,761 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 insecta_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ioPlaPenn1.1/dataset/CAKOGI01/snail.

Figure 5 .
Figure 5. Genome assembly of Platycnemis pennipes, ioPlaPenn1.1:Hi-C contact map of the ioPlaPenn1.1 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=HvZd4tspTCKewtlkoJJ4dQ.

Reviewer
Report 05 April 2024 https://doi.org/10.21956/wellcomeopenres.21899.r75670© 2024 Marcus 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.

Table 2 . Chromosomal pseudomolecules in the genome assembly of Platycnemis pennipes, ioPlaPenn1.
Table 3 contains a list of relevant software tool versions and sources.

Darwin Tree of Life Project Sampling Code of Practice', which
(Manni et al., 2021;Simão et al., 2015) 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.Surana et al., 2023b).The genome was analysed within the BlobToolKit environment(Challis et al., 2020)and BUSCO scores(Manni et al., 2021;Simão et al., 2015)were calculated.Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.The genome sequence is released openly for reuse.The Platycnemis pennipes 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.information Members of the Natural History Museum Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.4790042.Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.4893703.Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5013541.Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.