The genome sequence of a soldierfly, Nemotelus nigrinus (Fallén, 1817)

We present a genome assembly from an individual female Nemotelus nigrinus (a soldierfly; Arthropoda; Insecta; Diptera; Stratiomyidae). The genome sequence is 722.2 megabases in span. Most of the assembly is scaffolded into 5 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 18.94 kilobases in length.


Background
Nemotelus nigrinus is a small black fly (body length 3.25 to 4.5 mm) from the family Stratiomyidae (Diptera), commonly called soldierflies.It has a conical face characteristic for the genus, and is entirely black except for white halteres, and yellow parts of the legs (tips of femora, anterior and middle tibiae, parts of hind tibiae, tarsi).
The species is common in southern England, the East Midlands and the coastal belt of Wales, but less common in northern England, scarce in south-east Scotland, and there are a few scattered records from Ireland (Stubbs & Drake, 2014).The species occurs in fens and marsh on chalk and limestone, coastal marshes and around pools in the slacks of calcareous dunes.The adults are on the wing from May to August, with a peak in late June to early July (Stubbs & Drake, 2014).
The larva and puparium have not been described.The known Nemotelus larvae are associated with mud or surface of standing water (Rozkošný, 1982).A specimen of Nemotelus nigrinus has been reared from a swan's nest (Stubbs & Drake, 2014).
The high-quality genome of Nemotelus nigrinus 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 Nemotelus nigrinus, based on one female specimen from Cothill Fen National Nature Reserve, England.

Genome sequence report
The genome was sequenced from one female Nemotelus nigrinus (Figure 1) collected from Cothill Fen National Nature Reserve (51.69,.A total of 40-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 1,292 missing joins or mis-joins and removed 20 haplotypic duplications, reducing the assembly length by 0.62% and the scaffold number by 63.16%, and increasing the scaffold N50 by 181%. The final assembly has a total length of 722.2 Mb in 636 sequence scaffolds with a scaffold N50 of 217.6 Mb (Table 1).Most (98.15%) of the assembly sequence was assigned to 5 chromosomal-level scaffolds, representing 4 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).There are regions of unknown order and orientation in the following positions: Chromosome 1 (82,000 to 152,000 kbp, Chromosome 2 (100,000 to 135,000 kbp), Chromosome 3 (72,400 to 97,000 kbp) and Chromosome 4 (65,000 to 68,500 kbp).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/343700.

Sample acquisition and nucleic acid extraction
A female of Nemotelus nigrinus (idNemNigr1, NHMUK014036819) was swept using an insect net from the vegetation in Cothill Fen National Nature Reserve, England (51.69, -1.329) on 2021-06-19.The collector was Olga Sivell (Natural History Museum).The specimen was identified by  Duncan Sivell (Natural History Museum) using Stubbs and Drake (2014).The specimen was snap-frozen using dry ice.The tissue samples taken from it were stored in a CoolRack prior to genome sequencing.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The idNemNigr1 specimen was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Whole organism 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.

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 the Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from tissue of idNemNigr1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.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) DSL2 pipelines "sanger-tol/readmapping" (Surana et al., 2023a) and "sanger-tol/genomenote" (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.
Table 3 contains a list of relevant software tool versions and sources.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.
The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Software tool Version
Sivell and colleagues report the genome assembly of the soldier fly Nemotelus nigrinus.This is a thorough and organised genome report, with the use of appropriate methods and clear reporting, as is typical for assemblies that have come through the Darwin Tree of Life project pipelines.
I have just a few minor comments: (1) I suggest prefacing the collection site co-ordinates with latitude and longitude.
(2) The assembly contiguity statistics for this assembly are a little inferior to other assemblies I have seen come through this process (i.e.there are quite a large number of scaffolds, even after considerable manual curation).Were there particular features of the HiFi library that account for this?
(3) Do the regions of unknown order and orientation (one per autosome) share particular features/characteristics?

Hanno Schmidt
Johannes Gutenberg University Mainz, Mainz, Germany In the manuscript "The genome sequence of a soldierfly, Nemotelus nigrinus (Fallén, 1817)" by Olga Sivell et al. the authors present a genome assembly of a non-model organism fly.The assembly is based on PacBio HiFi reads and additional Hi-C data.The methods used are appropriate and the resulting assembly is at the chromosome level and of good quality.I would particularly like to emphasize the documentation of sampling and initial processing.This is sometimes presented a little too superficially in genome sequencing projects.Everything is traceable here, from sampling of the specimen, species identification, specimen identifiers and DNA extraction.A hyperlink provided in the manuscript leads to even more information on sample, sequencing and assembly.The text is concise, but clear and understandable.The genome data is publicly available.

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: genomics 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.Photographs of the Nemotelus nigrinus (specimen NHMUK014036819, individual idNemNigr1) taken during sample preservation and processing.A and B. A habitus of the specimen in dorsal view.C. The specimen in lateral view.

Figure 2 .
Figure 2. Genome assembly of Nemotelus nigrinus, idNemNigr1.1: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 722,201,843 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 (228,165,927 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (217,608,999 and 92,825,783 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 diptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idNemNigr1.1/dataset/CANBKX01/snail.

Figure 5 .
Figure 5. Genome assembly of Nemotelus nigrinus, idNemNigr1.1:Hi-C contact map of the idNemNigr1.1 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=RJt7Caf-RHia-GX3chzEJA.

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

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:
4) The Yahs pipeline (https://github.com/c-zhou/yahs)specifies several routes to read mapping, can you specify which one was used in this case?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.
https://doi.org/10.21956/wellcomeopenres.21638.r80586©2024 Schmidt H.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.