The genome sequence of the lesser worm flesh fly, Sarcophaga ( Sarcophaga) subvicina Rohdendorf, 1937

We present a genome assembly from an individual male Sarcophaga subvicina (the lesser worm flesh fly; Arthropoda; Insecta; Diptera; Sarcophagidae). The genome sequence is 71 megabases in span. Most of the assembly (95.91%) is scaffolded into six chromosomal pseudomolecules, with the X sex chromosome assembled. The mitochondrial genome has also been assembled and is 16.7 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,793 protein coding genes.


Background
Sarcophaga subvicina (Diptera: Sarcophagidae) is a relatively large (up to 8-15 mm (van Emden, 1954)) flesh fly with a Palearctic distribution (Pape, 1996).S. subvicina show the characteristic patterning of the Sarocophaga genus, with an overall blackish/greyish colouration, a checked abdomen, three longitudinal stripes on the thorax, and large red/orange eyes, and so can be difficult to separate from other members of the genus without examination of male genitalia or DNA barcoding (Jordaens et al., 2013;Szpila et al., 2015).Sarcophaga is a large genus, and the nearly 900 species contained within it are classified into 169 subgenera (Buenaventura et al., 2017), with S. subvicina placed in the Sarcophaga subgenus along with over 20 other species (Pape, 1996).The relative speciesrichness of this subgenus stands in stark contrast to the majority of sarcophagid subgenera, which are monotypic.The Sarcophaga subgenus contains three of the roughly 65 currently recognised UK Sarcophagid species (S. carnaria, S. variegata, and S. subvicina), in what is often termed the "carnaria group".
Sarcophaga subvicina is found across the UK, with a range that extends to the north of Scotland, and is most abundant between May and September (see: https://species.nbnatlas.org/species/NBNSYS0000030329).It has been reported as favouring open (urban/grassland) habitats (Fremdt & Amendt, 2014;Hwang & Turner, 2005), and adults have been attracted to large carcasses (Szpila et al., 2015).Larvae have been reported only from small mammal carcasses, and reared in captivity on meat and dead slugs (Blackith & Blackith, 1994;Pape, 1987), but this species seems to more likely represent an earthworm specialist.All Sarcophagids examined to date have a diploid chromosome number of 12, with an XY sex determination system and males the heterogametic sex (Srivastava & Gaur, 2015).
The genome of the lesser worm flesh fly S. subvicina 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 S. subvicina based on an individual male specimen from Wytham Woods, Berkshire.

Genome sequence report
The genome was sequenced from one male S. subvicina specimen collected in Wytham Woods, Berkshire (Figure 1).A total of 65-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 51-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 95 missing/misjoins and removed four haplotypic duplications, reducing the assembly length by 0.57% and the scaffold number by 16.97%, and increasing the scaffold N50 by 4.73%.
The final assembly has a total length of 714 Mb in 274 sequence scaffolds with a scaffold N50 of 123 Mb (Table 1).Most (95.91%) of the assembly sequence was assigned to six chromosomal-level scaffolds, representing 5 autosomes and the X sex chromosome (Figure 2-Figure 5; Table 2).Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size.This is a male specimen with known XY sex determination system, however we have been unable to identify Y sequences.The X chromosome is assembled from scaffolds of undetermined order and orientation.The assembly has a BUSCO 5.3. 2 (Manni et al., 2021) completeness of 99.2% (single 98.5%, duplicated 0.7%), using the diptera_odb10 reference set (n = 3,285).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.

Amendments from Version 1
The following changes have been made to the article: • The species taxonomic authority has been corrected to Rohdendorf, 1937 throughout.
• We have included a link to the TOLQC page providing assembly metadata for this sequencing project.
• We have added a chromosome grid to the Hi-C map for Figure 5.
• We have added information on species identification.
• We corrected the text on the sample homogenisation

Sample acquisition and nucleic acid extraction
A male S. subvicina (idSarSubv1) was collected and identified by Steven Falk (independent researcher).The species was identified using the latest keys to the identification of Sarcophagidae (https://osf.io/preprints/osf/vf5r6),and species identification was also confirmed by COI barcode.The specimen was collected using a net in Wytham Woods, Berkshire (latitude 51.766, longitude -1.309) and snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute.The idSarSubv1 sample was weighed and dissected on dry ice with head 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.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing.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 solidphase 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

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud 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) and Illumina NovaSeq 6000 (10X) instruments.Hi-C data were also generated from head tissue of idSarSubv1 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2021), which performed annotation using MitoFinder (Allio et al., 2020).The genome was analysed and BUSCO scores generated within the BlobToolKit environment (Challis et al., 2020).Table 3 contains a list of all software tool versions used, where appropriate.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) at the European Bioinformatics Institute (EBI) was used to generate annotation for the S. subvicina assembly (GCA_936449025.1).Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

Ethics/compliance issues
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the Darwin Tree of Life Project Sampling Code of Practice.By agreeing with and signing up to the Sampling Code of Practice,  freebayes (Garrison & Marth, 2012).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2022).The assembly was checked for contamination as described previously (Howe et al., 2021).Manual curation was performed using HiGlass (Kerpedjiev et al., 2018)  The genome sequence is released openly for reuse.The Sarcophaga subvicina 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.Raw data and assembly accession identifiers are reported in Table 1.

Andrzej Grzywacz
Nicolaus Copernicus University, Toruń, Poland The report describes the results of extraction, sequencing and assembly of genome of Sarcophaga subvicina.The protocols of molecular work are well described.Some remarks consider other aspects of this work.
An error is present in the title of this report.Sarcophaga (Sarcophaga) subvicina has been described by Rohdendorf, not by Baranov.The correct title should include: "Sarcophaga (Sarcophaga) subvicina Rohdendorf, 1937".
According to the catalogue of Pape (1996) it is a species of Palaearctic distribution.Please delete "Neararctic" [sic!].
Please correct cases where taxon names are not italicised.
Please provide some potential examples of studies where generated dataset can be employed.
I recommend adding more information concerning specimen identification.In particular, a reference to a taxonomic key used for species delimitation, i.e., latest species-level taxonomic literature that contains the currently accepted species concept (Meier 2017).
In the Methods section, please check whether a sentence " The only suggestion that I would like to make is that there be a paragraph included on the possible reasons why the Y chromosome sequences could not be identified as the authors already know that it should be present.
Once again, well done.

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 In this manuscript, the authors isolated nuclear and mitochondrial DNA from single male one male Sarcophaga subvicina and sequenced the DNA using Pacific Biosciences and 10X Genomics platforms, both of which generated long reads most appropriate for genome assembly due to their better ( 51-65) fold coverage than other technologies such as illimina generated short reads.
The assembly was successfully achieved to a high (chromosome) level followed by curation using an appropriate suite of bioinformatic tools and annotations based on transcriptomic data and protein from UniProt database.
The sample collection, DNA extraction, sequencing and assembly are well described and the results well presented.I however have the following concerns The importance of assembling this genome is not adequately presented, other than as part of a routine process in the as part of the Darwin Tree of Life Project and that that this species is attracted to large carcasses.Is there any other economic, medical/ veterinary etc importance of this species?What knowledge will better be understanding of the advance? 1.
While the transcriptomic data was used in the annotation of the genome, information on generation and nature of this data is missing.Was it from the same species?Was it tissue specific or whole body?The nature (source) of this data influences the gene families that will be preferentially annotated.For example, midgut derived RNA seq data will provide better annotation of midgut associated genes such as digestive genes than olfaction genes associated with the antennae.

2.
The assembly information would have been improved by information on genome arrangements such as synteny, intergenic sequences, transposon/repetitive sequence expansions, number of exons and their average size and orthologs in relation to closest relative among others.

3.
Information on nature of the genes annotated has not been provided.The information 4.
would yield important insights with broad implications important aspects of the flesh fly biology.The approach would identify gene families involved in important biological aspects of the fly such as its olfaction, flesh feeding/nutrition, immunity, microbiome, reproduction and developmental biology among other important aspects of this fly 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: Medical Entomology, Bioinformatics and 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, however I have significant reservations, as outlined above.

Figure 2 .
Figure 2. Genome assembly of Sarcophaga subvicina, idSarSubv1.1: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 711,151,016 bp assembly.The distribution of chromosome lengths is shown in dark grey with the plot radius scaled to the longest chromosome present in the assembly (159,501,612bp, shown in red).Orange and pale-orange arcs show the N50 and N90 chromosome lengths (132,242,496 and 118,606,681bp), respectively.The pale grey spiral shows the cumulative chromosome 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/idSarSubv1.1/dataset/CAKZFR01/snail.

Figure 3 .
Figure 3. Genome assembly of Sarcophaga subvicina, idSarSubv1.1:GC coverage.BlobToolKit GC-coverage plot.Chromosomes are coloured by phylum.Circles are sized in proportion to chromosome length.Histograms show the distribution of chromosome length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idSarSubv1.1/dataset/ CAKZFR01/blob.
spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Figure 4 .
Figure 4. Genome assembly of Sarcophaga subvicina, idSarSubv1.1:cumulative sequence.BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all chromosomes.Coloured lines show cumulative lengths of chromosomes assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idSarSubv1.1/dataset/CAKZFR01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Sarcophaga subvicina, idSarSubv1.1:Hi-C contact map.Hi-C contact map of the idSarSubv1.1 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=fjqWR98ySXisVhiBypHNoA.

Table 2 . Chromosomal pseudomolecules in the genome assembly of Sarcophaga subvicina, idSarSubv1. INSDC accession Chromosome Size (Mb) GC%
and Pretext 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.Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.

the rationale for creating the dataset(s) clearly described? No Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
Thorax tissue was [[if powermasher used: disrupted using a Nippi Powermasher fitted with a BioMasher pestle; else: cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts]]."is correct.No competing interests were disclosed.