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. 2024 Sep 18;9:533. [Version 1] doi: 10.12688/wellcomeopenres.23056.1

The genome sequence of the ruby bryozoan, Bugula neritina (Linnaeus, 1758)

Rebekka Uhl 1, John Bishop 1, Helen Jenkins 1, Christine Wood 1, Patrick Adkins 1, Freja Azzopardi 1; Marine Biological Association Genome Acquisition Lab; Darwin Tree of Life Barcoding collective; Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team; Wellcome Sanger Institute Scientific Operations: Sequencing Operations; Wellcome Sanger Institute Tree of Life Core Informatics team; Tree of Life Core Informatics collective; Darwin Tree of Life Consortiuma
PMCID: PMC11480708  PMID: 39415781

Abstract

We present a genome assembly from a specimen of Bugula neritina (the ruby bryozoan; Bryozoa; Gymnolaemata; Cheilostomatida; Bugulidae). The genome sequence has total length of 216.00 megabases. Most of the assembly is scaffolded into 9 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.25 kilobases in length. Gene annotation of this assembly on Ensembl identified 20,264 protein-coding genes.

Keywords: Bugula neritina, ruby bryozoan, genome sequence, chromosomal, Cheilostomatida

Species taxonomy

Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Spiralia; Lophotrochozoa; Bryozoa; Gymnolaemata; Cheilostomatida; Flustrina; Buguloidea; Bugulidae; Bugula; Bugula neritina (Linnaeus, 1758) (NCBI:txid10212).

Background

The erect colonies of the bryozoan Bugula neritina are commonly deep purplish-brown, and are formed of narrow, dichotomously dividing branches, two zooids wide ( Bilewitch, 2009; Hayward & Ryland, 1995). They could be mistaken for finely branched red seaweeds, but are much tougher in texture. The species was first described from the Mediterranean Sea. However, records of the bryozoan are widespread, ranging from New Zealand, the Atlantic and Gulf coast of America and Hawai’i. B. neritina is a highly successful fouling organism and is now also commonly found in the UK as an invasive species, especially within fouling communities ( Bilewitch, 2009; McGovern & Hellberg, 2003).

Molecular research has shown that B. neritina is in fact a complex of at least three cryptic species, Types S, D and N ( Davidson & Haygood, 1999). In addition to molecular characteristics, these species vary by habitat and geographical location. Type N (North Atlantic) and D (Deep) are mostly restricted to North America, whereas type S (Shallow) is widely distributed globally and is commonly found in the UK ( Fehlauer-Ale et al., 2014).

B. neritina was once thought to be the producer of bryostatins (cancer-fighting cyclic polyketides), but it has since been demonstrated that a bacterial symbiont Candidatus Endobugula sertula is responsible for bryostatin production ( Davidson et al., 2001). The production of these bryostatins is of ecological importance for the bryozoan as they are used to coat the larvae for protection ( Sharp et al., 2007). The presence of bryostatins in the coat has been found to be unpalatable to predators and thereby allows the larvae to settle successfully despite their large size and nutritional value ( Lindquist & Hay, 1996; McGovern & Hellberg, 2003). Interestingly, despite the importance of this bacterial symbiosis, type N B. neritina has only been seen to exhibit the symbiont when it is found further south of its usual distribution ( Linneman et al., 2014). This may be a consequence of lower predation pressure in the Northern range of Type N distribution; hence bryostatin production is less ecologically important ( McGovern & Hellberg, 2003).

Molecular research into these organisms will further provide an understanding of host-symbiont interactions, for example, by analysing the presence or absence of genes that control bryostatin production ( Hildebrand et al., 2004; Miller et al., 2016). A draft genome of B. neritina has previously been published (GCA_010799875.2) and used to better understand the evolution of bryozoans, for example via the expression of the Hox gene cluster ( Rayko et al., 2020; Saadi et al., 2023). Thus, the publication of the complete genome sequence of B. neritina expected to have a significant impact, particularly due to the pharmacological importance of bryostatins.

Genome sequence report

The genome of an adult Bugula neritina ( Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating a total of 27.37 Gb (gigabases) from 2.82 million reads, providing approximately 132-fold coverage. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data, which produced 85.23 Gbp from 564.45 million reads, yielding an approximate coverage of 395-fold. Specimen and sequencing information is summarised in Table 1.

Figure 1. Photograph of the Bugula neritina (tzBugNeri2) specimen used for genome sequencing.

Figure 1.

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Table 1. Specimen and sequencing data for Bugula neritina.

Project information
Study title Bugula neritina (ruby bryozoan)
Umbrella BioProject PRJEB66388
Species Bugula neritina
BioSample SAMEA112148689
NCBI taxonomy ID 10212
Specimen information
Technology ToLID BioSample accession Organism part
PacBio long read sequencing tzBugNeri2 SAMEA112152753 Modular colony
Hi-C sequencing tzBugNeri1 SAMEA7536669 Modular colony
Sequencing information
Platform Run accession Read count Base count (Gb)
Hi-C Illumina NovaSeq 6000 ERR12102386 5.64e+08 85.23
PacBio Sequel IIe ERR12085103 2.82e+06 27.37
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Manual assembly curation corrected 17 missing joins or mis-joins and one haplotypic duplications, reducing the assembly length by 6.39% and the scaffold number by 50.08%. The final assembly has a total length of 216.00 Mb in 320 sequence scaffolds, with 110 gaps, and a scaffold N50 of 24.8 Mb ( Table 2). 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 (92.7%) of the assembly sequence was assigned to 9 chromosomal-level scaffolds. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 5; Table 3). A large number of repetitive sequences could not be uniquely assigned to a chromosome. 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.

Figure 2. Genome assembly of Bugula neritina, tzBugNeri2.1: metrics.

Figure 2.

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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 216,009,554 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 (35,640,107 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (24,795,343 and 10,879,868 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/GCA_964035545.1/dataset/GCA_964035545.1/snail.

Figure 3. Genome assembly of Bugula neritina, tzBugNeri2.1: BlobToolKit GC-coverage plot.

Figure 3.

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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/GCA_964035545.1/dataset/GCA_964035545.1/blob.

Figure 4. Genome assembly of Bugula neritina tzBugNeri2.1: BlobToolKit cumulative sequence plot.

Figure 4.

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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/GCA_964035545.1/dataset/GCA_964035545.1/cumulative.

Figure 5. Genome assembly of Bugula neritina tzBugNeri2.1: Hi-C contact map of the tzBugNeri2.1 assembly, visualised using HiGlass.

Figure 5.

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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=EsDQ2q5VTHeLSFoZVP3Pag.

Table 2. Genome assembly data for Bugula neritina, tzBugNeri2.1.

Genome assembly
Assembly name tzBugNeri2.1
Assembly accession GCA_964035545.1
Accession of alternate haplotype GCA_964035465.1
Span (Mb) 216.00
Number of contigs 431
Contig N50 length (Mb) 4.0
Number of scaffolds 320
Scaffold N50 length (Mb) 24.8
Longest scaffold (Mb) 35.64
Assembly metrics * Benchmark
Consensus quality (QV) 53.7 ≥ 50
k-mer completeness 99.99% ≥ 95%
BUSCO ** C:83.4%[S:82.7%,D:0.7%],
F:7.5%,M:9.1%,n:954		 C ≥ 95%
Percentage of assembly mapped
to chromosomes		 92.7% ≥ 95%
Sex chromosomes None localised homologous pairs
Organelles Mitochondrial genome: 15.25 kb complete single alleles
Genome annotation of assembly GCA_964035545.1 at Ensembl
Number of protein-coding genes 20,264
Number of non-coding genes 1,222
Number of gene transcripts 33,722
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* Assembly metric benchmarks are adapted from column VGP-2020 of “Table 1: Proposed standards and metrics for defining genome assembly quality” from Rhie et al. (2021).

** BUSCO scores based on the metazoa_odb10 BUSCO set using version 5.4.3. 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/Bugula_neritina/dataset/GCA_964035545.1/busco.

Table 3. Chromosomal pseudomolecules in the genome assembly of Bugula neritina, tzBugNeri2.

INSDC accession Name Length (Mb) GC%
OZ037765.1 1 35.64 34.0
OZ037766.1 2 33.58 33.5
OZ037767.1 3 32.42 34.5
OZ037768.1 4 24.8 34.5
OZ037769.1 5 17.72 34.5
OZ037770.1 6 16.05 35.0
OZ037771.1 7 15.81 34.5
OZ037772.1 8 13.35 35.0
OZ037773.1 9 10.88 34.5
OZ037774.1 MT 0.02 29.0
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The estimated Quality Value (QV) of the final assembly is 53.7 with k-mer completeness of 99.99%, and the assembly has a BUSCO v5.4.3 completeness of 83.4% (single = 82.7%, duplicated = 0.7%), using the metazoa_odb10 reference set ( n = 954).

Metadata for specimens, BOLD barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/10212.

Genome annotation report

The Bugula neritina genome assembly (GCA_964035545.1) was annotated at the European Bioinformatics Institute (EBI) on Ensembl Rapid Release. The resulting annotation includes 33,722 transcribed mRNAs from 20,264 protein-coding and 1,222 non-coding genes ( Table 2; https://rapid.ensembl.org/Bugula_neritina_GCA_964035545.1/Info/Index). The average transcript length is 7,394.61. There are 1.57 coding transcripts per gene and 8.23 exons per transcript.

Methods

Sample acquisition

An adult specimen of Bugula neritina (specimen ID MBA-220815-011A, ToLID tzBugNeri2) was collected from the Mayflower Marina, Plymouth, Devon, UK (latitude 50.36, longitude

–4.17) on 2022-08-15. The specimen was collected by Patrick Adkins, Freja Azzopardi and Rebekka Uhl and identified by John Bishop (all from the Marine Biological Association) and then preserved on dry ice.

The specimen used for Hi-C sequencing (specimen ID MBA-200715-001A, ToLID tzBugNeri1) was an adult specimen collected from Queen Anne's Battery Marina Visitors' Pontoon, Plymouth, Devon, UK (latitude 50.36, longitude –4.13) on 2020-07-15. The specimen was collected and identified by Patrick Adkins, John Bishop and Christine Wood, and preserved by liquid nitrogen.

The initial identification was verified by an additional DNA barcoding process according to the framework developed by Twyford et al. (2024). A small sample was dissected from the specimens and stored in ethanol, while the remaining parts of the specimen were shipped on dry ice to the Wellcome Sanger Institute (WSI). The tissue was lysed, the COI marker region was amplified by PCR, and amplicons were sequenced and compared to the BOLD database, confirming the species identification ( Crowley et al., 2023). Following whole genome sequence generation, the relevant DNA barcode region was also used alongside the initial barcoding data for sample tracking at the WSI ( Twyford et al., 2024). The standard operating procedures for Darwin Tree of Life barcoding have been deposited on protocols.io ( Beasley et al., 2023).

Nucleic acid extraction

The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation and homogenisation, DNA extraction, fragmentation and purification. Detailed protocols are available on protocols.io ( Denton et al., 2023b). The tzBugNeri2 sample was weighed and dissected on dry ice ( Jay et al., 2023). Tissue from the modular colony was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a). HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol ( Oatley et al., 2023). The DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system ( Bates et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation, using AMPure PB beads to eliminate shorter fragments and concentrate the DNA ( Strickland et al., 2023). The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

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 IIe instrument.

Hi-C data were generated from frozen tissue from tzBugNeri1 using the Arima-HiC v2 kit. In brief, frozen tissue (–80 °C) was fixed, and the DNA crosslinked using a TC buffer containing formaldehyde. The crosslinked DNA was then digested using a restriction enzyme master mix. The 5’-overhangs were then filled in and labelled with a biotinylated nucleotide and proximally ligated. The biotinylated DNA construct was fragmented to a fragment size of 400 to 600 bp using a Covaris E220 sonicator. The DNA was then enriched, barcoded, and amplified using the NEBNext Ultra II DNA Library Prep Kit, following manufacturers’ instructions. The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on an Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation

Assembly

The HiFi reads were first assembled using Hifiasm ( Cheng et al., 2021) with the --primary option. Haplotypic duplications were identified and removed using purge_dups ( Guan et al., 2020). The Hi-C reads were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019). The contigs were further scaffolded using the provided Hi-C data ( Rao et al., 2014) in YaHS ( Zhou et al., 2023) using the --break option. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al., 2022), BUSCO ( Manni et al., 2021) and MERQURY.FK ( Rhie et al., 2020).

The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Assembly curation

The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants (ASCC) pipeline (article in preparation). Flat files and maps used in curation were generated in TreeVal ( Pointon et al., 2023). Manual curation was primarily conducted using PretextView ( Harry, 2022), with additional insights provided by JBrowse2 ( Diesh et al., 2023) and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Any identified contamination, missed joins, and mis-joins were corrected, and duplicate sequences were tagged and removed. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation (article in preparation).

Evaluation of the final assembly

The final assembly was post-processed and evaluated using the three Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines: sanger-tol/readmapping ( Surana et al., 2023a), sanger-tol/genomenote ( Surana et al., 2023b), and sanger-tol/blobtoolkit ( Muffato et al., 2024). The readmapping pipeline aligns the Hi-C reads using bwa-mem2 ( Vasimuddin et al., 2019) and combines the alignment files with SAMtools ( Danecek et al., 2021). The genomenote pipeline converts the Hi-C alignments into a contact map using BEDTools ( Quinlan & Hall, 2010) and the Cooler tool suite ( Abdennur & Mirny, 2020). The contact map is visualised in HiGlass ( Kerpedjiev et al., 2018). This pipeline also generates assembly statistics using the NCBI datasets report ( Sayers et al., 2024), computes k-mer completeness and QV consensus quality values with FastK and MERQURY.FK, and runs BUSCO ( Manni et al., 2021) to assess completeness.

The blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline ( Challis et al., 2020). It aligns the PacBio reads in SAMtools and minimap2 ( Li, 2018) and generates coverage tracks for regions of fixed size. In parallel, it queries the GoaT database ( Challis et al., 2023) to identify all matching BUSCO lineages to run BUSCO ( Manni et al., 2021). For the three domain-level BUSCO lineages, the pipeline aligns the BUSCO genes to the UniProt Reference Proteomes database ( Bateman et al., 2023) with DIAMOND ( Buchfink et al., 2021) blastp. The genome is also split into chunks according to the density of the BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database with DIAMOND blastx. Genome sequences without a hit are chunked with seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). The blobtools suite combines all these outputs into a blobdir for visualisation.

The genome assembly and evaluation pipelines were developed using nf-core tooling ( Ewels et al., 2020) and MultiQC ( Ewels et al., 2016), relying on the Conda package manager, the Bioconda initiative ( Grüning et al., 2018), the Biocontainers infrastructure ( da Veiga Leprevost et al., 2017), as well as the Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017) containerisation solutions.

Table 4 contains a list of relevant software tool versions and sources.

Table 4. Software tools: versions and sources.

Software tool Version Source
BEDTools 2.30.0 https://github.com/arq5x/bedtools2
BLAST 2.14.0 ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/
BlobToolKit 4.3.7 https://github.com/blobtoolkit/blobtoolkit
BUSCO 5.4.3 and 5.5.0 https://gitlab.com/ezlab/busco
bwa-mem2 2.2.1 https://github.com/bwa-mem2/bwa-mem2
Cooler 0.8.11 https://github.com/open2c/cooler
DIAMOND 2.1.8 https://github.com/bbuchfink/diamond
fasta_windows 0.2.4 https://github.com/tolkit/fasta_windows
FastK 427104ea91c78c3b8b8b49f1a7d6bbeaa869ba1c https://github.com/thegenemyers/FASTK
Gfastats 1.3.6 https://github.com/vgl-hub/gfastats
GoaT CLI 0.2.5 https://github.com/genomehubs/goat-cli
Hifiasm 0.19.8-r603 https://github.com/chhylp123/hifiasm
HiGlass 44086069ee7d4d3f6f3f0012569789ec138f42b84
aa44357826c0b6753eb28de		 https://github.com/higlass/higlass
Merqury.FK d00d98157618f4e8d1a9190026b19b471055b
22e		 https://github.com/thegenemyers/MERQURY.FK
MitoHiFi 3 https://github.com/marcelauliano/MitoHiFi
MultiQC 1.14, 1.17, and 1.18 https://github.com/MultiQC/MultiQC
NCBI Datasets 15.12.0 https://github.com/ncbi/datasets
Nextflow 23.04.0-5857 https://github.com/nextflow-io/nextflow
PretextView 0.2 https://github.com/sanger-tol/PretextView
purge_dups 1.2.5 https://github.com/dfguan/purge_dups
samtools 1.16.1, 1.17, and 1.18 https://github.com/samtools/samtools
sanger-tol/ascc - https://github.com/sanger-tol/ascc
sanger-tol/
genomenote		 1.1.1 https://github.com/sanger-tol/genomenote
sanger-tol/
readmapping		 1.2.1 https://github.com/sanger-tol/readmapping
Seqtk 1.3 https://github.com/lh3/seqtk
Singularity 3.9.0 https://github.com/sylabs/singularity
TreeVal 1.0.0 https://github.com/sanger-tol/treeval
YaHS 1.2a.2 https://github.com/c-zhou/yahs
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Genome annotation

The Ensembl Genebuild annotation system ( Aken et al., 2016) was used to generate annotation for the Bugula neritina assembly (GCA_964035545.1) 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

•   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, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.

Funding Statement

This work was supported by Wellcome through core funding to the Wellcome Sanger Institute [206194, <a href=https://doi.org/10.35802/206194>https://doi.org/10.35802/206194</a>] and the Darwin Tree of Life Discretionary Award [218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328 </a>].

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[version 1; peer review: 1 approved, 2 approved with reservations]

Data availability

European Nucleotide Archive: Bugula neritina (ruby bryozoan). Accession number PRJEB66388; https://identifiers.org/ena.embl/PRJEB66388 ( Wellcome Sanger Institute, 2023). The genome sequence is released openly for reuse. The Bugula neritina 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 and Table 2.

Author information

Members of the Marine Biological Association Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.8382513.

Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.12158331

Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team are listed here: https://doi.org/10.5281/zenodo.12162482.

Members of Wellcome Sanger Institute Scientific Operations: Sequencing Operations are listed here: https://doi.org/10.5281/zenodo.12165051.

Members of the Wellcome Sanger Institute Tree of Life Core Informatics team are listed here: https://doi.org/10.5281/zenodo.12160324.

Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.12205391.

Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.

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Wellcome Open Res. 2024 Oct 15. doi: 10.21956/wellcomeopenres.25392.r103511

Reviewer response for version 1

Ferdinand Marlétaz 1

This reports described the sequencing and assembly of the bryozoan,  Bugula neritina at chromsome scale. This is a welcome dataset for a group of organisms that is sometimes overlooked. Probably because of the type I genome architecture of bryozoan (see Hoencamp 2021), the HiC contact map is really difficult to read and does not bring much. As I usually comment, it would be great to have more details about HiC procedures. It is good to have an annotation, maybe specificy its Busco would be also interesting.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

genomics, zoology, phylogeny

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.

Wellcome Open Res. 2024 Oct 12. doi: 10.21956/wellcomeopenres.25392.r102194

Reviewer response for version 1

Emily Giles 1,2

The authors report a genome assembly and annotation for Bugula neritina. They used PacBio, HiFi and HiC to generate a high quality assembly mapped to 9 pseudochromosomes and including 320 scaffolds. A primary assembly was generated using Hifiasm, haplotypic duplications were purged with purge_dups, then HiC reads were mapped to the primary assembly for scaffolding. The assembly was annotated using the Ensembl genebuild pipeline. In general, the manuscript is well written and clear, though the methods are not always detailed in the manuscript directly rather specification is supplied via links which requires the reader to search elsewhere for details.

Overall, the results are robust, but I do have a few concerns/questions:

  1. Please provide a bit more information in the methods so that the reader does not have to go to your github page for details, i.e. curation process and blobtools methods.

  2. Different specimens were used for the PacBio and HiC libraries. What was the rational for this? How would this affect the assembly? This should be explained.

  3. The authors briefly describe their assembly curation and evaluation process, but it is not clear if contamination identified with blobtools was removed from the assembly, i.e does the assembly contain Mollusca, Brachiopoda, and Cnidaria?

  4.  The assembly has decent BUSCO scores with the metazoan database, though complete BUSCOs are only at 84.8%. This could be due to the evolutionary history of the species/ composition of metazoan database skewed towards other lineages, but given that the annotation does not include ab initio prediction, many genes could have been missed in the annotation process. This should be stated somewhere. Furthermore, regarding the annotation pipeline, all cDNAs from ENA and RefSeq were used to predict cDNAs? Or a subset of transcriptomes was used? This is not clear.

Overall, these resources will be valuable for evolutionary studies in Bryozoa. It would be nice for non-Bryozoan experts to know how these resources contribute to currently available genomic resources for Bryozoan or other closely related taxa.

Are sufficient details of methods and materials provided to allow replication by others?

Partly

Is the rationale for creating the dataset(s) clearly described?

Partly

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Comparative genomics, genome assembly, genome annotation, marine genomics, transcriptomics, population 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.

Wellcome Open Res. 2024 Oct 9. doi: 10.21956/wellcomeopenres.25392.r100213

Reviewer response for version 1

Tim Yue Him Wong 1

This work reports the chromosome level assembly of B. neritina tzBugNeri2.1 which is based on 132x PacBio data and Arima2 Hi-C data. The assembly process included initial PacBio assembly generation , haplotig separation, and Hi-C based scaffolding. The mitochondrial genome was also assembled and reported. 

While the authors spend a full paragraph in the introduction mentioning bryostatin and host-symbiont relationship, this work included no information about the symbiont genome or any potential bacterial genomic fragments (as a side product from Hi-C based scaffolding) were probably excluded as noise. 

In my opinion, this work, in addition to host-symbiont interaction, is very important to the study of evodevo, in terms of understanding of the evolution of bryozoan, especially this work finally provided a comprehensive understanding on the list and structure of important developmental genes such as HOX, signaling pathway genes such as Wnt, BMP, Shh etc (Wong et al. 2014; Fuchs et al. 2011). This will eventually enable the accurate positioning of bryozoan (Ectoprocta) in the lophotrochozoan and provide novel insight into evolution of spiralian, such as the development of body axis. I think this can be further elaborate in the final paragraph of the introduction where you can include citation on B. neritina developmental gene studies.

For the method part, I am concerned by the condition of the specimen, because the specimen in the photo in Figure 1 has brownish color while a healthy colony, should have dark purple color. I checked the method part and there were no content on the assessment of the DNA quality. Also, it said in the Genome annotation part that " Annotation was created primarily through alignment of transcriptomic data to the genome", yet none was mentioned on the origin of the transcritpome data (RNAseq? use existing RNAseq dataset in SRA?). If the authors used the specimen shown in Figure 1 to generate a transcriptome dataset, I think the gene annotation part may inevitably have bias toward a subset of genes relating to cell-death or genes relating to degradation.

Are sufficient details of methods and materials provided to allow replication by others?

Partly

Is the rationale for creating the dataset(s) clearly described?

Partly

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Partly

Reviewer Expertise:

Single cell RNAseq, evo-devo, eco-devo, larval settlement and metamorphosis, IsoSeq, dark proteome

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.

References

  • 1. : Gene expression in bryozoan larvae suggest a fundamental importance of pre-patterned blastemic cells in the bryozoan life-cycle. Evodevo .2011;2(1) : 10.1186/2041-9139-2-13 13 10.1186/2041-9139-2-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Citations

    1. Wellcome Sanger Institute: The genome sequence of the ruby bryozoan, Bugula neritina (Linnaeus, 1758). European Nucleotide Archive.[dataset], accession number PRJEB66388,2023.

    Data Availability Statement

    European Nucleotide Archive: Bugula neritina (ruby bryozoan). Accession number PRJEB66388; https://identifiers.org/ena.embl/PRJEB66388 ( Wellcome Sanger Institute, 2023). The genome sequence is released openly for reuse. The Bugula neritina 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 and Table 2.

    Articles from Wellcome Open Research are provided here courtesy of The Wellcome Trust

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