The genome sequence of the Brown Litter Worm, Bimastos eiseni (Levinsen, 1884)

We present a genome assembly from an individual Bimastos eiseni (the Brown Litter Worm; Annelida; None; Haplotaxida; Lumbricidae). The genome sequence is 660.5 megabases in span. Most of the assembly is scaffolded into 17 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.34 kilobases in length.


Background
Bimastos eiseni ( (Figure 1) is identified using external morphological features, including the presence of a tanylobic head alongside a clitellum located on segments 24-27 to 32--33 and no visible tubercula pubertatis (Sherlock, 2018).Genetic studies have shown that this species is most likely North American in origin (though there are very few published records of this species on GBIF, GBIF Secretariat, 2024) and it should be considered non-native in Europe (Csuzdi et al., 2017).Despite this, B. eiseni is listed on the Global List of Introduced and Invasive Species Register for the USA and not on the lists for European territories where it is known to occur.B. eiseni has been recorded in England, Scotland and Ireland, but not yet recorded from Wales or Northern Ireland.The distribution is thought to be widespread through most of England, with sparse records in northern England, Scotland and Wales.
It is an epigeic (surface-dwelling) species of earthworm that is often associated with deadwood microhabitats, including tree hollows.It contributes towards the decomposition of waste plant material, contributing to waste recycling and detoxification ecosystem services (Keith & Robinson, 2012).
We present a chromosomally complete genome sequence for Bimastos eiseni, based on one specimen from Wytham Woods, Oxfordshire, UK, as part of the Darwin Tree of Life Project.This project is a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.

Genome sequence report
The genome was sequenced from a specimen of Bimastos eiseni collected from Wytham Woods, Oxfordshire, UK (51.78, -1.34).A total of 32-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 98 missing joins or mis-joins and removed 8 haplotypic duplications, reducing the scaffold number by 24.42%.
The final assembly has a total length of 660.5 Mb in 129 sequence scaffolds with a scaffold N50 of 41.3 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 (99.66%) of the assembly sequence was assigned to 17 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).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.

Sample acquisition and nucleic acid extraction
Specimens of Bimastos eiseni were collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.78, longitude -1.34) on 2021-05-27.The specimens were collected and identified by Keiron Brown (University of Oxford), with confirmation of species by Emma Sherlock (Natural History Museum), and snap-frozen on dry ice.A specimen with ID Ox001367 (ToLID whEisEise2) was used for DNA sequencing and a specimen with ID Ox001365 (ToLID whEisEise1) was used for Hi-C sequencing.
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.In sample preparation, the whEisEise2 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue from the posterior body was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).
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

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 II instrument.Hi-C data were also generated from posterior body tissue of whEisEise1 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 contamination and corrected as described previously (Howe et al., 2021).Manual curation was performed using HiGlass    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 • Legality of collection, transfer and use (national and international) 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,  Given the diversity in earthworm sizes, it would be beneficial to include a scale bar in Figure 1 to provide clearer context.Additionally, once genome annotation becomes available, it would be valuable for the authors to comment on the mitochondrial genome, noting any significant differences between it and the whole genome.This could offer interesting insights and further enrich the study.

Dong Zhang
1 Tibet University, Lhasa, China 2 Lanzhou University, Lanzhou, Gansu, China The paper is well-written, with a high-quality genome that is thoroughly analyzed and presented.The methodology is sound, and I found the Methods section particularly informative.However, I have one concern: the genome appears to lack structural annotation.Completing the annotation work would greatly enhance the manuscript and should be considered before indexing.
Is the rationale for creating the dataset(s) clearly described?

José M Martín-Durán
Queen Mary University of London, London, UK This genome note reports the sequencing and assembly of the genome of the earthworm Bimastos eiseni, a species thought to be invasive in the US but that might actually come from there and be non-native in Europe.Bimastos eiseni is an important ecological player, as it is often associated with the decomposition of organic matter in soils.A high-quality genome will help with elucidating the origin of this species and studies on their ecological role.
Strengths: High-quality genome assembly and important resource for a potentially invasive species.

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Weaknesses: Taxonomical category "None" in the abstract should be amended.This belongs to Clitellata.

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Correct typo in the second line of the background (extra -between 32 and 33).

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Annotation not available, but this will be sorted in the future.Reviewer Expertise: Evolutionary biology, developmental biology, comparative 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.Photograph of a preserved specimen of Bimastos eiseni (not the specimen used for genome sequencing) taken by Keiron Derek Brown and Kerry Calloway.

Figure 2 .
Figure 2. Genome assembly of Bimastos eiseni, whEisEise2.1: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 660,529,080 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 (71,778,175 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (41,312,700 and 23,769,983 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 metazoa_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/whEisEise2_1/dataset/whEisEise2_1/snail.

Figure 3 .
Figure 3. Genome assembly of Bimastos eiseni, whEisEise2.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/whEisEise2_1/dataset/whEisEise2_1/blob.

Figure 4 .
Figure 4. Genome assembly of Bimastos eiseni, whEisEise2.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/whEisEise2_1/dataset/whEisEise2_1/cumulative.
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.

Figure 5 .
Figure 5. Genome assembly of Bimastos eiseni, whEisEise2.1:Hi-C contact map of the whEisEise2.1 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=eyFDKRI1QIeJbPX-y2JIPg.

©
2024 Tilikj N.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.Natasha Tilikj Complutense University of Madrid, Madrid, Spain Brown et al. have submitted a manuscript detailing the genomic assembly of the Brown Litter Worm, an epigeic species with a broad distribution across much of England.Using long-read sequencing and Hi-C data, they have produced a genome of exceptional quality, as demonstrated by the assembly metrics.Although my expertise in genome conformation techniques is somewhat limited, I found the results and the chromosomal visualization in Figure 5 to be clearly presented and easy to follow.

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Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.

Table 1 . Genome data for Bimastos eiseni, whEisEise2.1. Project accession data
(Strickland et al., 2023)rks are adapted from column VGP-2020 of "Table1: Proposed standards and metrics for defining genome assembly quality" from(Rhie et al., 2021).(Stricklandetal., 2023): 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 and 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(Dentonetal., 2023b).
Table 3 contains a list of relevant software tool versions and sources.
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

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.

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.

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: genome 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.
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.