Abstract
We present a genome assembly from an individual Lineus longissimus (the bootlace worm; Nemertea; Pilidiophora; Heteronemertea; Lineidae). The genome sequence is 391 megabases in span. The majority of the assembly is scaffolded into 19 chromosomal pseudomolecules.
Keywords: Lineus longissimus, bootlace worm, genome sequence, chromosomal
Species taxonomy
Eukaryota; Metazoa; Spiralia; Lophotrochozoa; Nemertea; Pilidiophora; Heteronemertea; Lineidae; Lineus; Lineus longissimus (Gunnerus, 1770) (NCBI:txid88925).
Introduction
Lineus longissimus (Lineidae, Heteronemertea, Nemertea) is a predatory ribbon worm, renowned as being the longest animal in the British and Irish biota, as individual specimens can exceed 25 m when fully extended. Their extensibility results in part from their unsegmented body form, where the coleom is limited to a rhynchocoel associated with the eversible proboscis. Phylum Nemertea is placed as sister to either Mollusca or Annelida within Eutrochozoa in the Spiralia ( Struck & Fisse, 2008). Nemertea includes only 1200 described species worldwide that play important ecological roles in littoral and benthic communities ( Gibson, 1972). Nemerteans have been studied for their ability to regenerate body parts ( Zattara et al., 2019) and the potent venom neurotoxins secreted from the glandular epithelium of the proboscis ( Stricker & Cloney, 1983). The nemertide alpha-1 toxin from L. longissimus shows promise as an insecticide ( Bell et al., 2021).
Genome sequence report
The genome was sequenced from a single L. longissimus of unknown sex collected from White Bay, Great Cumbrae, North Ayreshire, Scotland (latitude 55.790409, longitude -4.908826). A total of 79-fold coverage in Pacific Biosciences single-molecule long reads and 107-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 67 missing/misjoins and removed 5 haplotypic duplications, reducing the assembly length by 0.46% and the scaffold number by 63.10%, and increasing the scaffold N50 by 53.76%. The final assembly has a total length of 391 Mb in 32 sequence scaffolds with a scaffold N50 of 21 Mb ( Table 1). Of the assembly sequence, 99.81% was assigned to 19 chromosomal-level scaffolds, representing 19 autosomes (numbered by sequence length) ( Figure 1– Figure 4; Table 2). The assembly has a BUSCO ( Simão et al., 2015) v5.1.2 completeness of 96.5% using the metazoa_odb10 reference set. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.
Table 1. Genome data for Lineus longissimus, tnLinLong1.1.
| Project accession data | |
|---|---|
| Assembly identifier | tnLinLong1.1 |
| Species | Lineus longissimus |
| Specimen | tnLinLong1 |
| NCBI taxonomy ID | NCBI:txid88925 |
| BioProject | PRJEB45185 |
| BioSample ID | SAMEA7522833 |
| Isolate information | Unknown sex, anterior/mid/
posterior body |
| Raw data accessions | |
| PacificBiosciences
SEQUEL II |
ERR6412039, ERR6436382,
ERR6436383 |
| 10X Genomics Illumina | ERR6054915-ERR6054918 |
| Hi-C Illumina | ERR6054914 |
| Genome assembly | |
| Assembly accession | GCA_910592395.1 |
| Accession of alternate haplotype | GCA_910592375.1 |
| Span (Mb) | 391 |
| Number of contigs | 109 |
| Contig N50 length (Mb) | 10 |
| Number of scaffolds | 32 |
| Scaffold N50 length (Mb) | 21 |
| Longest scaffold (Mb) | 29 |
| BUSCO * genome score | C:96.5%[S:96.1%,D:0.4%],F:2.2%,M:1.3%,n:954 |
*BUSCO scores based on the metazoa_odb10 BUSCO set using v5.1.2. C= complete [S= single copy, D=duplicated], F=fragmented, M=missing, n=number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/tnLinLong1.1/dataset/CAJUZJ01/busco.
Figure 1. Genome assembly of Lineus longissimus, tnLinLong1.1: metrics.
The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/tnLinLong1.1/dataset/CAJUZJ01/snail.
Figure 2. Genome assembly of Lineus longissimus, tnLinLong1.1: GC coverage.
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/tnLinLong1.1/dataset/CAJUZJ01/blob. It should be noted that the tnLinLong1 genome is the first of its phylum (Nemertea) to be assembled, meaning there are few/no other related sequences for BlobToolKit to call in INSDC databases. The resultant identification of Mollusca, Chordata and Brachiopoda sequences here reflects the divergence of Nemertea from other phyla.
Figure 3. Genome assembly of Lineus longissimus, tnLinLong1.1: 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. Since the tnLinLong1 genome is the first of its phylum (Nemertea) to be assembled, there are few/no other related sequences for BlobToolKit to call in INSDC databases. The resultant identification of Mollusca, Chordata and Brachiopoda sequences here reflects the divergence of Nemertea from other phyla. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/tnLinLong1.1/dataset/CAJUZJ01/cumulative.
Figure 4. Genome assembly of Lineus longissimus, tnLinLong1.1: Hi-C contact map.
Hi-C contact map of the tnLinLong1.1 assembly, visualised in HiGlass.
Table 2. Chromosomal pseudomolecules in the genome assembly of Lineus longissimus, tnLinLong1.1.
| INSDC
accession |
Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| OU342992.1 | 1 | 29.45 | 41.0 |
| OU342993.1 | 2 | 29.19 | 41.1 |
| OU342994.1 | 3 | 29.08 | 41.2 |
| OU342995.1 | 4 | 25.60 | 41.2 |
| OU342996.1 | 5 | 23.94 | 41.3 |
| OU342997.1 | 6 | 21.66 | 41.8 |
| OU342998.1 | 7 | 21.16 | 41.3 |
| OU342999.1 | 8 | 20.96 | 41.3 |
| OU343000.1 | 9 | 20.55 | 41.4 |
| OU343001.1 | 10 | 19.41 | 41.4 |
| OU343002.1 | 11 | 19.19 | 41.5 |
| OU343003.1 | 12 | 17.98 | 41.6 |
| OU343004.1 | 13 | 16.85 | 41.6 |
| OU343005.1 | 14 | 16.76 | 41.5 |
| OU343006.1 | 15 | 16.29 | 42.0 |
| OU343007.1 | 16 | 16.53 | 41.5 |
| OU343008.1 | 17 | 16.30 | 41.6 |
| OU343009.1 | 18 | 15.89 | 41.4 |
| OU343010.1 | 19 | 13.63 | 41.7 |
| OU343011.1 | MT | 0.02 | 35.3 |
| - | Unplaced | 0.74 | 42.5 |
Methods
A single L. longissimus of unknown sex was collected from White Bay, Great Cumbrae, North Ayreshire, Scotland (latitude 55.790409, longitude -4.908826) by Dominic Kwiatkowski, Wellcome Sanger Institute (WSI) and preserved on dry ice prior to transfer to the WSI.
DNA was extracted from midbody tissue at the Tree of Life laboratory, WSI. The tnLinLong1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Samples were cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts. Fragment size analysis of 0.01-0.5 ng of DNA was then performed using an Agilent FemtoPulse. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. Low molecular weight DNA was removed from a 200-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 between 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.
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud sequencing libraries were constructed according to the manufacturers’ instructions. Sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II and Illumina HiSeq X instruments. Hi-C data were generated using the Arima v2.0 kit and sequenced on HiSeq X.
Assembly was carried out with Hifiasm ( Cheng et al., 2021), haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). The assembly was polished with the 10X Genomics Illumina data by aligning to the assembly with longranger align, calling variants with freebayes ( Garrison & Marth, 2012). One round of the Illumina polishing was applied. Scaffolding with Hi-C data ( Rao et al., 2014) was carried out with SALSA2 ( Ghurye et al., 2019). The assembly was checked for contamination and corrected using the gEVAL system ( Chow et al., 2016) as described previously ( Howe et al., 2021). Manual curation was performed using gEVAL, HiGlass ( Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2021). 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.
Table 3. Software tools used.
| Software tool | Version | Source |
|---|---|---|
| Hifiasm | 0.12 | Cheng et al., 2021 |
| purge_dups | 1.2.3 | Guan et al., 2020 |
| longranger | 2.2.2 |
https://support.10xgenomics.com/genome-exome/software/pipelines/latest/advanced/
other-pipelines |
| freebayes | v1.3.1-17-
gaa2ace8 |
Garrison & Marth, 2012 |
| MitoHiFi | 2.0 | Uliano-Silva et al., 2021 |
| SALSA2 | 2.2 | Ghurye et al., 2019 |
| gEVAL | N/A | Chow et al., 2016 |
| HiGlass | 1.11.6 | Kerpedjiev et al., 2018 |
| PretextView | 0.2.x | https://github.com/wtsi-hpag/PretextView |
| BlobToolKit | 2.6.2 | Challis et al., 2020 |
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, 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.
Data availability
European Nucleotide Archive: Lineus longissimus (bootlace worm). Accession number PRJEB45185: https://identifiers.org/ena.embl:PRJEB45185
The genome sequence is released openly for reuse. The L. longissimus 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. The genome will be annotated and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.
Acknowledgments
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.4893704.
Members of the Wellcome Sanger Institute Tree of Life programme collective are listed here: https://doi.org/10.5281/zenodo.5377053.
Members of Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective are listed here: https://doi.org/10.5281/zenodo.4790456.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5013542.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783559.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (206194) and the Darwin Tree of Life Discretionary Award (218328).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 1; peer review: 2 approved]
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