The genome sequence of a braconid wasp, Aleiodes leptofemur van Achterberg & Shaw, 2016

We present a genome assembly from an individual male Aleiodes leptofemur (braconid wasp; Arthropoda; Insecta; Hymenoptera; Braconidae). The genome sequence spans 271.20 megabases. Most of the assembly is scaffolded into 15 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 32.28 kilobases in length.


Background
Aleiodes leptofemur is a small (about 4.5 mm long) braconid wasp of the subfamily Rogadinae.One of many similar species of Aleiodes, A. leptofemur can be identified using van Achterberg and Shaw (2016); useful features for distinguishing A. leptofemur from other dark and reddish-brown Aleiodes include the particularly slender femora and colour details of face, antenna and hind leg.
As with all other Rogadinae, where known, the hosts are Lepidoptera larvae which are mummified by the wasp larva, forming a hardened structure in which the wasp pupates (Zaldívar-Riverón et al., 2008).A wide range of Noctuidae are attacked, with the common factor being that they feed low down in vegetation.The winter is spent as a larva in the host which is often manipulated to climb to an exposed position before being killed.This habit means that A. leptofemur is frequently reared from the exposed mummies.The adults are partly nocturnal and often light-trapped.
Found across much of Europe, A. leptofemur has multiple generations per year and adults are long-lived (van Achterberg & Shaw, 2016).Until relatively recently, specimens had been misidentified as other Aleiodes, especially A. borealis (Thomson); however, van Achterberg and Shaw (2016) established that this species had been undescribed and formally described A. leptofemur, including extensive data on the ecology of this widespread species.This genome will help in deciphering the diversification of this very species-rich genus, including testing hypotheses of host range evolution (Shaw, 2002).

Genome sequence report
The genome of an adult male Aleiodes leptofemur (Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating a total of 14.67 Gb (gigabases) from 1.40 million reads, providing approximately 59-fold coverage.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data, which produced 116.45 Gbp from 771.22 million reads, yielding an approximate coverage of 429-fold.Specimen and sequencing information is summarised in Table 1.
Manual assembly curation corrected 87 missing joins or mis-joins, reducing the scaffold number by 66.67%.The final assembly has a total length of 271.20 Mb in 16 sequence scaffolds with a scaffold N50 of 17.3 Mb (Table 2).The total count of gaps in the scaffolds is 263.The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.97%) 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 5; Table 3).The specimen was a haploid male.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition
An adult male Aleiodes leptofemur (specimen ID NHMUK010636352, ToLID iyAleLepo1) was collected from Tonbridge, England, UK (latitude 51.19, longitude 0.26) on 2021-05-28 using a light trap.The specimen was collected and identified by Gavin Broad (Natural History Museum), and was preserved by dry freezing at -80 °C.
Following morphological identification, the species taxonomy is verified by DNA barcoding according to the framework developed by Twyford et al. (2024).Briefly, legs from the specimen are taken and stored in ethanol.The tissue is lysed, the CO1 gene is amplified by PCR, and amplicons are sequenced and compared to the BOLD database (Crowley et al., 2023).A DNA barcode is also generated from the PacBio sequencing data at a later stage for sample tracking through the genome production pipeline at the Wellcome Sanger Institute (Twyford et al., 2024).The standard operating procedures for the Darwin Tree of Life barcoding have been deposited on protocols.io(Beasley et al., 2023).

Nucleic acid extraction
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 iyAleLepo1 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue from the whole organism was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).
HMW DNA was extracted at the WSI Scientific Operations core using the Automated MagAttract v2 protocol (Oatley et al., 2023).The DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 31 (Bates et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023): in brief, the method employs AMPure PB beads 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 using the 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 remaining whole organism tissue of iyAleLepo1 using the Arima-HiC v2 kit.The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on the Illumina NovaSeq 6000 instrument.

Assembly curation
The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants (ASCC) pipeline (article in preparation).Flat files and maps used in curation were generated in TreeVal (Pointon et al., 2023).Manual curation was primarily conducted using PretextView (Harry, 2022), with additional insights provided by JBrowse2 (Diesh et al., 2023) and HiGlass (Kerpedjiev et al., 2018).Scaffolds were visually inspected and corrected as described by Howe et al. (2021).Any identified contamination, missed joins, and mis-joins were corrected, and duplicate sequences were tagged and removed.The entire process is documented at https://gitlab.com/wtsi-grit/rapid-curation (article in preparation).

Evaluation of the final assembly
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  The genome assembly and evaluation pipelines were developed using the nf-core tooling (Ewels et al., 2020), use MultiQC (Ewels et al., 2016), and make extensive use of the Conda package manager, the Bioconda initiative (Grüning et al., 2018), the Biocontainers infrastructure (da Veiga Leprevost et al., 2017), and the Docker (Merkel, 2014) and Singularity (Kurtzer et al., 2017) containerisation solutions.subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree 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

Software tool Version
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

Julien Varaldi
Universite Claude Bernard Lyon 1, Villeurbanne, France This manuscript presents the complete chromosome-level assembly of Aleiodes leptofemur (braconid wasp; Arthropoda; Insecta; Hymenoptera; Braconidae).This is a parasitoid wasp that attacks Lepidoptera caterpillars.The manuscript is clear and concise and the analysis is sound.The final assembly is of high quality thanks to the use of short and long reads and HiC data obtained from the DNA of a single (haploid) male.

I have only very minor comments:
I think the peculiar way of life (parasitoid) of this species is not clearly stated in the introduction.A few additional details on its biology could also be useful to the reader.For instance is it an endo-or an ecto-parasitoid?a solitary or gregarious parasitoid?It would also have been nice to know why the authors focused on this particular species.

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Please provide a reference for this assertion, if available."The winter is spent as a larva in the host which is often manipulated to climb to an exposed position before being killed."

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There is a bit of inconsistency in the coverage values provided in the result section.

Andrew D Austin
The University of Adelaide, Adelaide, South Australia, Australia This is a very competent piece of research which presents a fully annotated genome of a braconid wasp.As such it will be highly useful for a plethora of future studies on the biology and evolution of the group.The methods are well articulated and easy to follow, and the data clearly presented.
I have but two suggestions to improve the paper: First a little more detail about the characters helpful for identifying the subfamily and genus would assist anyone not familiar with the group. 1.
Second, a statement at the end of the introduction regarding the rationale for choosing this particular genus/species AND the significance of this genome for guiding future studies would be useful.My understanding is that there are very few fully annotated genomes for braconid wasps, and so this study is of particular importance.

2.
Is the rationale for creating the dataset(s) clearly described?Partly 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: molecular phylogenetics, evolution and systematics of parasitic wasps 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 Aleiodes leptofemur, iyAleLepo1.1: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 271,235,516 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 (26,012,348 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (17,287,625 and 15,370,087 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 hymenoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Aleiodes_leptofemur/dataset/GCA_963942555.1/snail.

Figure 3 .
Figure 3. Genome assembly of Aleiodes leptofemur, iyAleLepo1.1: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/Aleiodes_leptofemur/dataset/GCA_963942555.1/blob.

Figure 4 .
Figure 4. Genome assembly of Aleiodes leptofemur iyAleLepo1.1: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/Aleiodes_leptofemur/dataset/GCA_963942555.1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Aleiodes leptofemur iyAleLepo1.1:Hi-C contact map of the iyAleLepo1.1 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=ci1GSod4RD-2U_QcFn3hKA.

Table 4
contains a list of relevant software tool versions and sources.
have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is

Open Peer Review Current Peer Review Status: Version 1
Wellcome Sanger Institute, 2024).The genome sequence is released openly for reuse.The Aleiodes leptofemur 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 and Table 2.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.

the rationale for creating the dataset(s) clearly described? Partly 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:
It is mentioned that 14.67Gb of PacBio reads have been produced, giving a 59-fold coverage: The genome is thus expected to be 14.67/59 = 0.2486441 Gb.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.
Reviewer Report 18 September 2024 https://doi.org/10.21956/wellcomeopenres.25073.r96678© 2024 Austin A. 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.