The genome sequence of the Banded Burying beetle, Nicrophorus investigator Zetterstedt, 1824

We present a genome assembly from a female Nicrophorus investigator (Banded Burying beetle; Arthropoda; Insecta; Coleoptera; Silphidae). The genome sequence is 202.3 megabases in span. Most of the assembly is scaffolded into 7 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 23.3 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,046 protein coding genes.


Background
The Banded Burying beetle Nicrophorus investigator Zetterstedt, 1824 is a beetle in the Silphidae family.Species in the genus Nicrophorus are commonly called "Burying Beetles" due to their ability to transport and bury small vertebrate carcasses, a tactic deployed to avoid competition from other scavengers (Milne & Milne, 1944;Scott, 1998).Burying beetles are unusual in that they display bi-parental care of the offspring, rearing them underground in the carcass.This behaviour has been intensively studied (Pukowski, 1933;Smiseth & Moore, 2004;Trumbo, 1996;Trumbo, 2006).This species can be recognised by its large size (length 12-22 mm), striking orange and black markings on the elytra and the club of the antennae mostly orange (Duff, 2012;Fowler, 1889;Lane et al., 2021).Some individuals can occur with the orange frontal band of the elytra narrowed and interrupted near the suture, sometimes causing confusion with Nicrophorus interruptus.The presence of dark fringing hairs on all but the terminal abdominal tergite, help to differentiate this species from N. interruptus, which has golden fringing hairs on all abdominal tergites.
Like other carrion beetles, this species plays an important role in the decomposition of vertebrate remains and is therefore also important in forensic entomology, as its presence on a corpse can help to establish the minimum post-mortem interval (Amendt et al., 2004).
N. investigator is found in both forests and open habitats and has been recorded from a wide variety of carrion, with peak activity between July-September.Like most of the other Burying beetles, it is univoltine, but what sets it apart from most other Nicrophorus in the UK is that it breeds in late summer through to mid-autumn.The offspring overwinter as pre-pupae, completing their development in late Spring.Adults are largely crepuscular, and frequently appear in light traps (Lane, 2020).
It is a common and widely distributed species in Britain and Ireland, with an IUCN status of Least Concern (Lane, 2020).Its global distribution encompasses much of Europe, western Asia, reaching further east through Siberia to Japan and China (Růžička, 2015).It is also present in North America, in northern and western mountainous areas (Sikes, 2005).
The Nicrophorus investigator genome has not been sequenced previously.The genome and methylome of a related species Nicrophorus vespilloides have been generated (Cunningham et al., 2015).The high-quality chromosomal-level genome sequence described here, has been generated as part of the Darwin Tree of Life project.It will aid research into the taxonomy, biology and ecology of the species, and possible forensic applications.

Genome sequence report
The genome was sequenced from one female Nicrophorus investigator (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 91-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 19 missing joins or mis-joins and removed 5 haplotypic duplications, reducing the assembly length by 0.50% and the scaffold number by 2.29%, and increasing the scaffold N50 by 72.68%. The final assembly has a total length of 202.3 Mb in 127 sequence scaffolds with a scaffold N50 of 28.4 Mb (Table 1).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 (92.51%) of the assembly sequence was assigned to 7 chromosomal-level scaffolds, representing 6 autosomes and the X sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).Chromosome X was assigned based on synteny to Phosphuga atrata (GCA_944588485.1)(Crowley et al., 2023).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.

Genome annotation report
The Nicrophorus investigator genome assembly (GCA_ 963457615.In sample preparation, the icNicInve2 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue from the thorax was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).

Sample acquisition and nucleic acid extraction
HMW DNA was extracted using the Automated MagAttract v1 protocol (Sheerin et al., 2023).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., 2023): in brief, the method employs  contamination and corrected using the TreeVal rapid pipeline (Pointon et al., 2023).Manual curation was performed using JBrowse2 (Diesh et al., 2023), HiGlass (Kerpedjiev et al., 2018) and PretextView (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The Ensembl Genebuild annotation system (Aken et al., 2016) was used to generate annotation for the Nicrophorus investigator assembly (GCA_963457615.1) in Ensembl Rapid Release at the EBI.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).

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 of materials by a Darwin Tree of Life Partner is 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 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 RNA was extracted from thorax tissue of icNicInve2 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
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.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences Sequel IIe (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from thorax tissue of icNicInve2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 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 then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for

Zhonghua Wei
China West Normal University, Nanchong, China The high-quality chromosomal-level genome sequence of Nicrophorus investigator was described, which will aid research into the taxonomy, biology and ecology of the species, and possible forensic applications.The mitogenome of this species was also assembled in this study.
The format of this manuscript is very good and can be accepted for publication.The authors mention that the assembly is not fully phased but they deposited a second haplotype, which can help distinguish haplotypes.Overall, the manuscript is well written and the data has been presented nicely.This assembly is fully relevant and is significant because of the species' role in decomposition.
This study can be a valuable resource for further research in comparative genomics and forensic entomology.
Is the rationale for creating the dataset(s) clearly described?

Manee M Manee
King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia The study presents the first genome assembly of the Banded Burying beetle, Nicrophorus investigator.The assembly achieved a BUSCO score of 99.2%, indicating a high level of completeness and accuracy.The study identified 11,046 protein-coding genes and 1,790 noncoding genes through gene annotation.
This genomic data aims to support further research into the taxonomy, biology, and ecology of the species, as well as potential forensic applications, given the beetle's role in decomposition processes.The study's findings contribute valuable genetic resources for understanding the evolutionary traits and ecological roles of Nicrophorus investigator.I am happy with the manuscript in its current form.

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: bioinformatics, genomics and evolutionary biology 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 Nicrophorus investigator, icNicInve2.1: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 202,291,714 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 (39,255,461 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (28,407,949 and 13,674,797 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 endopterygota_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAUOPU01/dataset/CAUOPU01/snail.

Figure 3 .
Figure 3. Genome assembly of Nicrophorus investigator, icNicInve2.1: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/CAUOPU01/dataset/CAUOPU01/blob.
icNicInve2) was collected from Wytham Woods (latitude 51.77, longitude -1.31) on 2021-09-02 using an aerial net.The specimen was collected by Liam Crowley (University of Oxford) and Gavin Broad, Chris Fletcher, Inez Januszczak and Ian Barnes (Natural History Museum), identified by Liam Crowley, and preserved by dry freezing at -80 °C.The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.

Figure 5 .
Figure 5. Genome assembly of Nicrophorus investigator, icNicInve2.1:Hi-C contact map of the icNicInve2.1 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=cVAeHo4eS9GKimP1Azethg.

Figure 4 .
Figure 4. Genome assembly of Nicrophorus investigator, icNicInve2.1: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/CAUOPU01/dataset/CAUOPU01/cumulative.

©
2024 Bollepogu Raja K.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.Komal Kumar Bollepogu Raja Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA Crowley et al. present the genome assembly of Nicrophorus investigator in this article.The genome assembly is high quality and spans 202.3 Mb and includes 7 chromosomal pseudomolecules.The authors have extensively annotated the genome with 11,046 protein-coding genes.The strengths of this study are the high-quality assembly, detailed annotation and methodological rigor.The methods have been well documented, and the study can be reproduced based on the methods supplied in the text.

Table 2 . Chromosomal pseudomolecules in the genome assembly of Nicrophorus investigator, icNicInve2. INSDC accession Chromosome Length (Mb) GC%
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 and Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

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.

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.

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.