The genome sequence of the Coccidian parasite, Eimeria praecox (Apicomplexa: Eucoccidiorida)

We present a genome assembly from sporozoites from a clonal line of Eimeria praecox (the Coccidian parasite; Apicomplexa; Conoidasida; Eucoccidiorida; Eimeriidae). The genome sequence is 64.3 megabases in span. Most of the assembly is scaffolded into 15 chromosomal pseudomolecules. The organelle genomes have also been assembled and the mitochondrial genome is 6.23 kilobases in length, while the apicoplast genome is 28.83 kilobases long.


Background
Eimeria praecox is one of ten Eimeria species known to infect Gallus gallus domesticus (Blake et al., 2021).Widely regarded as mildly pathogenic, high level E. praecox infection can result in malabsorptive coccidiosis in chickens defined by failure to thrive and reduced bodyweight gain (Williams et al., 2009).The E. praecox Houghton strain was isolated at the Houghton Poultry Research Station (HPRS) in 1966 from a faecal sample collected from chickens in the west of Scotland (Shirley et al., 1984).Serving as a reference type isolate for the species, a clonal line derived from the Houghton strain was maintained at the Institute for Animal Health Compton site following the closure of HPRS and is now curated at the Royal Veterinary College, Hertfordshire, UK.Here, we present a chromosomally-complete genome sequence assembly constructed as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in Britain and Ireland.

Genome sequence report
The genome was sequenced from purified sporozoites from a clonal population of Eimeria praecox.A total of 476-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 55 missing joins or mis-joins and removed 10 haplotypic duplications, reducing the assembly length by 1.23% and the scaffold number by 48.57%, and increasing the scaffold N50 by 17.82%.
The final assembly has a total length of 64.3 Mb in 16 sequence scaffolds with a scaffold N50 of 5.2 Mb (Table 1).The snail plot in Figure 1 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 2. The cumulative assembly plot in Figure 3 shows curves for subsets of scaffolds assigned to different phyla.Most (99.92%) of the assembly sequence was assigned to 15 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 4; Table 2).Although Eimeria praecox is expected to be clonal, there was sufficient heterozygosity to separate an apparent alternate haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial and plastid genomes were also assembled and can be found as contigs within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
Oocysts of a clonal E. praecox line were harvested from experimentally infected G. gallus domesticus at the Royal Veterinary College, Hawkshead Campus, Hertfordshire, UK. Specific pathogen free (SPF) Lohmann Valo chickens were infected by oral inoculation of sporulated E. praecox Houghton strain oocysts and progeny oocysts were recovered four to six days later as described previously (Long et al., 1976).Oocysts were sporulated and processed to hatch and purify sporozoites following standard methods using nylon wool and DE-52 columns (Pastor-Fernández et al., 2019).Animals were raised in strict accordance with the Animals (Scientific Procedures) Act 1986, an Act of Parliament of the United Kingdom.All animal studies and protocols were approved by the Royal Veterinary College Animal Welfare & Ethical Review Body (AWERB) and the UK Government Home Office under project licence.The collected sample had specimen ID SAN0001685 and ToLID pxEimPrae1.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the pxEim-Prae1 sample was weighed and aliquoted on dry ice (Jay et al., 2023), and the cell pellet was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).HMW DNA was extracted using the Manual MagAttract v1 protocol (Strickland et al., 2023b).DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023a): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer with a Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

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 a Pacific Biosciences Sequel IIe instrument.Hi-C data were also generated from pxEim-Prae1 using the Arima2 kit.The library was made using a Hi-C-Arima v2 kit with restriction enzyme motif ^GATC,G ^ANTC,C^TNAG,T^TAA.The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on the Illumina NovaSeq 6000 instrument.

Final assembly evaluation
The final assembly was post-processed and evaluated with the three Nextflow (Di Tommaso et al  The sanger-tol/blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline (Challis et al., 2020).It aligns the PacBio reads with SAMtools and minimap2 (Li, 2018) and generates coverage tracks for regions of fixed size.In parallel, it queries the GoaT database (Challis et al., 2023) to identify all matching BUSCO lineages to run BUSCO (Manni et al., 2021).For the three domain-level BUSCO lineage, the pipeline aligns the BUSCO genes to the Uniprot  et al., 1990).All those outputs are combined with the blobtools suite into a blobdir for visualisation.
All three pipelines were developed using the nf-core tooling (Ewels et al., 2020), use MultiQC (Ewels et al., 2016), and make 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 Darwin Tree of Life Partner.The submission 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 data note presents a chromosome level assembly for a clonal line of Eimeria praecox, a pathogen originally isolated from poultry and maintained in culture since 1966.Despite the expected clonal growth in culture, an alternate haplotype was also discovered.The sequencing quality and coverage obtained appear excellent, and BUSCO score indicates no missing genes.The source material, sequencing and bioinformatics methods are described in good detail.

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: Parasitology, evolution, speciation
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The manuscript presents the genome assembly of the Houghton strain of Eimeria praecox.More specifically it reports on a reference type isolate, which was originally collected in Scotland in 1966 and is now maintained at the Royal Veterinary College as a reference type isolate for the species.
The rationale for generating a genome sequence for this important isolate is clear.
A clear and concise report summaries the state-of-the art techniques used for the project.The wet-lab techniques and bioinformatics pipelines are clearly described and (especially the latter, for which it is easier to judge) fulfill modern standards of reproducibility.
The manuscript presents quality metrics which illustrate the (high) quality of the chromosomallycomplete genome sequence.The dataset is available through generally established openly accessible databases in standardized format usable for anyone with the relevant knowledge and skills.
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: Parasitology, Genomics, Transcriptomics, 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 1 .
Figure 1.Genome assembly of Eimeria praecox, pxEimPrae1.1:metrics.The BlobToolKit snail plot 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 64,286,523 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 (9,795,849 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (5,197,822 and 2,647,507 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 coccidia_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Eimeria_praecox/dataset/GCA_963920595.1/snail.

Figure 2 .
Figure 2. Genome assembly of Eimeria praecox, pxEimPrae1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Eimeria_praecox/dataset/GCA_963920595.1/blob.

Figure 3 .
Figure 3. Genome assembly of Eimeria praecox, pxEimPrae1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Eimeria_praecox/dataset/GCA_963920595.1/cumulative.

Figure 4 .
Figure 4. Genome assembly of Eimeria praecox, pxEimPrae1.1:Hi-C contact map of the pxEimPrae1.1 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=er1InEPwTtyeAh7mNn1xYA.

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:
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.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.