The genome sequence of the red compost earthworm, Lumbricus rubellus (Hoffmeister, 1843)

We present a genome assembly from an individual Lumbricus rubellus (the red compost earthworm; Annelida; Clitellata; Haplotaxida; Lumbricidae). The genome sequence is 787.5 megabases in span. Most of the assembly is scaffolded into 18 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.81 kilobases in length. Gene annotation of this assembly on Ensembl identified 33,426 protein coding genes.

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Background
Lumbricus rubellus (Hoffmeister, 1843) is an earthworm that feeds on decaying organic matter near the soil surface.Up to 130 mm in length, it has a cylindrical body in cross section except for a flattened posterior, possessing a purplish pigmentation dorsally at the head-end (James, 2010) (Figure 1).Though native to Europe, L. rubellus has become an invasive species through accidental and deliberate transport worldwide (James, 2010;Klein et al., 2020).Typical of lumbricid species, L. rubellus exhibits highly divergent mitochondrial lineages, with evidence of five distinct lineages across Europe (Giska et al., 2015).However, in the UK, just two (lineages A and B) are found, a reduced diversity likely due to limited re-colonisation after the loss of the land bridge to continental Europe following glacial retreat (Jones et al., 2016).Despite this divergence, the mitochondrial lineages are not entirely reproductively isolated (Giska et al., 2015), even though reproductive pheromone variation and different habitat preferences reinforce lineage separation (Jones et al., 2016;Spurgeon et al., 2016).The genome presented here represents the A lineage, which appears to be the more dominant of the two lineages in the UK (Spurgeon et al., 2016).
Renowned ecologist John Stewart Collis described earthworms as "Eyeless, legless, faceless, voiceless, the earth-worm is not much to look at -a mere squirming piece of flesh," yet capable of "remarkable works".The genome of L. rubellus, a litter-inhabiting and cosmopolitan species, will provide insights into their particular abilities, including their unique metabolism, potential for tissue regeneration, as well as their capacity to colonise soils with varying contamination profiles and dramatic proton concentration differences.The relevance of L. rubellus to ecotoxicology (Morgan et al., 2004), ecology (Uvarov, 2009), biotechnology (Bakar et al., 2011), and evolutionary biology (Ferrier, 2012) makes this genome a vital resource for a broad range of scientific disciplines.
We present the complete genome sequence of Lumbricus rubellus, an earthworm species collected from Dinas Powys in south Wales 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 one Lumbricus rubellus from a culture collection held at the Kille Lab, University of Cardiff.A total of 30-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 36-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 459 missing joins or misjoins and removed 358 haplotypic duplications, reducing the assembly length by 14.03% and the scaffold number by 51.72%, and increasing the scaffold N50 by 0.15%.
The final assembly has a total length of 787.5 Mb in 380 sequence scaffolds with a scaffold N50 of 41.4 Mb (Table 1).Most (98.53%) of the assembly sequence was assigned to 18 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-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.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found on the Wellcome Sanger Tree of Life website.

Sample acquisition and nucleic acid extraction
The Lumbricus rubellus specimens used for genome sequencing (specimen ID SAN0001201, individual wcLumRube1), Hi-C scaffolding (specimen ID SAN0001205, wcLumRube5) and RNA sequencing (specimen ID SAN0001202, wcLum-Rube2) were obtained from a culture collection held at UKCEH, Wallingford, United Kingdom on 2020-03-17.This culture was generated (and regularly supplemented) using L. rubellus collected from Dinas Powys, Wales, United Kingdom (51.44, -3.24).The specimens were collected by Stephen Short, Amaia Green Etxabe and Alex Robinson (UK Centre for Ecology and Hydrology).The specimens were identified by Stephen Short and then flash frozen in liquid nitrogen.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The wcLumRube1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Bodywall 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 the 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 solid-phase reversible immobilisation using AMPure PB beads with a 1.8X RNA was extracted from body wall tissue of wcLumRube2 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud 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 II (HiFi), Illumina HiSeq 4000 (RNA-Seq) and HiSeq X Ten (10X) instruments.Hi-C data were also generated from body wall tissue of wcLumRube1 and wcLumRube5 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 and HiSeq X Ten instruments.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with    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 gene annotation system (Aken et al., 2016) was used to generate annotation for the Lumbricus rubellus assembly (GCA_945859605.1).Annotation was

Yoshiaki Morino
Institute of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan This paper reports the genome sequence of the red compost earthworm Lumbricus rubellus.The authors constructed a genome assembly using Pacific Biosciences HiFi long reads, 10X Genomics reads, and Hi-C data.Most of these sequences are mapped to 18 chromosome-level scaffolds.The mitochondrial genome was also assembled.The characteristics of this species, its research history, and the rationale for genome sequencing are clearly described.The quality of the assembly is high enough that I believe this genomic data will be a valuable resource for ecology, evolutionary biology and biotechnology.One minor suggestion for the authors is to introduce that this species (or earthworms in general) are hermaphrodites, possibly related to the lack of sex chromosomes.
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: Marine invertebrate, Evo-Devo 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.
responses or comments on the article can be found at the end of the article.

Figure 2 .
Figure 2. Genome assembly of Lumbricus rubellus, wcLumRube1.1: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 787,530,981 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 (68,399,915 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (41,365,854 and 33,209,857 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/Lumbricus%20rubellus/dataset/CAMAOG01/snail.

Figure 5 .
Figure 5. Genome assembly of Lumbricus rubellus, wcLumRube1.1:Hi-C contact map of the wcLumRube1.1 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=O4FKa6EsSKmCY1sc7Gjldw.

Table 3 . Software tools: versions and sources. Software tool Version 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).

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.

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