The genome sequence of the fish leech, Piscicola geometra (Linnaeus, 1761)

We present a genome assembly from an individual Piscicola geometra (the fish leech; Annelida; Clitellata; Hirudinida; Piscicolidae). The genome sequence is 171.1 megabases in span. Most of the assembly is scaffolded into 17 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.1 kilobases in length.


Background
Piscicola geometra, the fish leech, is an ectoparasite of freshwater fish.It is widespread in the UK, inhabiting well-oxygenated rivers and still waters that support its fish hosts.In lake Windermere the species completes its lifecycle within 7 to 9 months, allowing for 2 to 3 generations per year (Elliott & Dobson, 2015).
Piscicolid leeches have large anterior and posterior suckers that are clearly distinct from the rest of the body, which readily separates them from other British leech families.Until 2012, Piscicola geometra was assumed to be the sole species of Piscicola in Britain.However, Piscicola specimens collected in Yorkshire in 2006 were determined to be Piscicola siddalli, which was described as a new species in 2012 by Bielecki et al.Consequently, it is likely that some British records of Piscicola geometra might actually pertain to P. sidalli.As of March 2023, the Environment Agency has records from 194 distinct survey sites encompassing most English counties for Piscicola siddalli, compared to 4384 distinct survey sites for P. geometra (data source: (Environment Agency, 2021)).
Identifying mature, clearly marked specimens of P. geometra and P. siddalii is straightforward: dorsally P. geometra has a large, central diamond shaped spot of pale pigment on most body segments (Figure 1), compared to 3 to 5 transverse, small, pale spots in P. siddalli.Furthermore, there are differences in the number of subdivisions in the body segments, and the body shape, but these features are subtle and can be difficult to interpret (Bielecki et al., 2012;Elliott & Dobson, 2015).
Here we present a chromosomally complete genome sequence for Piscicola geometra, sequenced as part of the Darwin Tree of Life project, based on one specimen collected from Gloucester and Sharpness Canal, England.

Genome sequence report
The genome was sequenced from an individual Piscicola geometra (Figure 1) collected from Gloucester and Sharpness Canal, England, UK (latitude 51.86, longitude -2.26).A total of 76-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 43 missing joins or mis-joins and removed 6 haplotypic duplications, reducing the assembly length by 0.61%% and the scaffold number by 15.85%.
The final assembly has a total length of 171.1 Mb in 69 sequence scaffolds with a scaffold N50 of 9.3 Mb (Table 1).Most (96.08%) of the assembly sequence was assigned to 17 chromosomal-level scaffolds.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/60958.

Sample acquisition and nucleic acid extraction
A Piscicola geometra (specimen no, NHMUK014361516, individual wrPisGeom1) was collected from Gloucester and Sharpness Canal, England, UK (latitude 51.86, longitude -2.26) on 19 March 2019.The specimen was taken from freshwater using a kicknet, and then snap-frozen on dry ice.The   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.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from tissue of wrPisGeom1 that had been set aside, using the Arima2 kit and sequenced on the HiSeq X Ten 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 scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination as described previously (Howe et al., 2021).Manual curation was 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 Tommaso et al., 2017) et al., 2021;Simão et al., 2015) were calculated.
Table 3 contains a list of relevant software tool versions and sources.

Legal and ethical review process for Darwin Tree of Life Partner submitted materials
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.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 • Legality of collection, transfer and use (national and international) Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer

Software tool Version
Strengths: -High-quality chromosome-level genome assembly generated with gold-standard approaches.
-As an important fish parasite, this is a species that has an ecological and economic impact. Weaknesses: -The genome has no annotation.It might be helpful to highlight this will come soon.

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, invertebrate evolutionary and developmental biology,

Qingyou Liu
Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan, Guangdong, China DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from tissue of wrPisGeom1 that had been set aside, using the Arima2 kit and sequenced on the HiSeq X Ten instrument.The methods used in this article are suitable.
The manuscript is well written, interesting and meaningful.The fish leech genome is useful.The genome sequence is 171.1 megabases in span.Most of the assembly is scaffolded into 17 chromosomal pseudomolecules.The mitochondrial genome has also been assembled and is 15.1 kilobases in length.It can be Approved.

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: Animal genome and functional genome 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 Piscicola geometra, wrPisGeom1.1: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 171,095,250 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 (16,636,273 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (9,301,023 and 6,573,335 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 metazoa_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/wrPisGeom1.1/dataset/ CALSEQ01/snail.

Figure 5 .
Figure 5. Genome assembly of Piscicola geometra, wrPisGeom1.1:Hi-C contact map of the wrPisGeom1.1 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=Ye2V71QxQ3qnmuGmSHkfRA.

Table 1 . Genome data for Piscicola geometra, wrPisGeom1.1. Project accession data
(Rhie et al., 2021)enchmarks are adapted from column VGP-2020 of "Table1: Proposed standards and metrics for defining genome assembly quality" from(Rhie et al., 2021).The tissue was prepared and DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The wrPisGeom1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Whole organism 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