The genome sequence of the highfin lizardfish, Bathysaurus mollis Günther, 1878 (Aulopiformes: Bathysauridae)

TL;DR

This paper provides the genome sequence of the highfin lizardfish, a deep-sea fish, as part of a larger project to sequence species in Britain and Ireland.

Contribution

The novel contribution is the first reference genome assembly for the highfin lizardfish, including chromosomal scaffolding and mitochondrial genome.

Findings

The genome assembly has a total length of 1,065.77 megabases.

95.16% of the assembly is organized into 24 chromosomal pseudomolecules.

The mitochondrial genome is 16.68 kilobases in length.

Abstract

We present a genome assembly from an individual
Bathysaurus mollis (highfin lizardfish; Chordata; Actinopteri; Aulopiformes; Bathysauridae). The genome sequence has a total length of 1 065.77 megabases. Most of the assembly (95.16%) is scaffolded into 24 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, with a length of 16.68 kilobases. This assembly was generated as part of the Darwin Tree of Life project, which produces reference genomes for eukaryotic species found in Britain and Ireland.

Figures

Photographs of the Bathysaurus mollis Günther, 1878 specimen (DISCOLL_JC231_082_023; fBatMol1) used for genome sequencing.Top: lateral view of whole specimen on board prior to tissue sampling and preservation. Bottom: Dorsal detail view of head of the same specimen.

Frequency distribution of k-mers generated using GenomeScope2.The plot shows observed and modelled k-mer spectra, providing estimates of genome size, heterozygosity, and repeat content based on unassembled sequencing reads.

Hi-C contact map of the Bathysaurus mollis genome assembly.Assembled chromosomes are shown in order of size and labelled along the axes, with a megabase scale shown below. The plot was generated using PretextSnapshot.

Evaluation of k-mer completeness using MerquryFK.This plot illustrates the recovery of k-mers from the original read data in the final assemblies. The horizontal axis represents k-mer multiplicity, and the vertical axis shows the number of k-mers. The black curve represents k-mers that appear in the reads but are not assembled. The green curve corresponds to k-mers shared by both haplotypes, and the red and blue curves show k-mers found only in one of the haplotypes.

Assembly metrics for fBatMol1.1.The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1 000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the actinopterygii_odb10 set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer.

BlobToolKit GC-coverage plot for fBatMol1.1.Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available on the BlobToolKit viewer.

Tables

Table 1.. Specimen and sequencing data for BioProject PRJEB76377.

Platform	PacBio HiFi	Hi-C	RNA-seq
ToLID	fBatMol1	fBatMol1	fBatMol1
Specimen ID	NHMUK014453704	NHMUK014453704	NHMUK014453704
BioSample (source individual)	SAMEA114806302	SAMEA114806302	SAMEA114806302
BioSample (tissue)	SAMEA114806440	SAMEA114806440	SAMEA114806441
Tissue	muscle	muscle	muscle
Instrument	Revio	Illumina NovaSeq X	Illumina NovaSeq X
Run accessions	ERR13245303	ERR13248980	ERR14379122
Read count total	2.56 million	589.11 million	74.82 million
Base count total	27.55 Gb	88.96 Gb	11.30 Gb

Table 2.. Genome assembly statistics.

Assembly name	fBatMol1.1
Assembly accession	GCA_965279225.1
Alternate haplotype accession	GCA_965279235.1
Assembly level	chromosome
Span (Mb)	1 065.77
Number of chromosomes	24
Number of contigs	5 733
Contig N50	0.41 Mb
Number of scaffolds	1 095
Scaffold N50	44.26 Mb
Organelles	Mitochondrion: 16.68 kb

Table 3.. Chromosomal pseudomolecules in the primary genome assembly of Bathysaurus mollis fBatMol1.

INSDC accession	Molecule	Length (Mb)	GC%
OZ257213.1	1	57.87	42
OZ257214.1	2	48.43	42
OZ257215.1	3	47.95	42
OZ257216.1	4	47.73	42
OZ257217.1	5	47.39	41.50
OZ257218.1	6	47.35	42
OZ257219.1	7	46.57	42.50
OZ257220.1	8	45.69	42
OZ257221.1	9	45.12	42.50
OZ257222.1	10	44.92	42
OZ257223.1	11	44.45	42
OZ257224.1	12	44.26	42
OZ257225.1	13	43.56	41.50
OZ257226.1	14	43.09	42.50
OZ257227.1	15	42.90	42
OZ257228.1	16	42.75	42
OZ257229.1	17	40.41	42.50
OZ257230.1	18	39.78	42.50
OZ257231.1	19	39.28	42
OZ257232.1	20	36.96	42.50
OZ257233.1	21	35.59	42.50
OZ257234.1	22	29.91	42.50
OZ257235.1	23	27.71	42
OZ257236.1	24	24.49	42.50

Table 4.. Earth Biogenome Project summary metrics for the Bathysaurus mollis assembly.

Measure	Value	Benchmark
EBP summary (primary)	5.C.Q50	6.C.Q40
Contig N50 length	0.41 Mb	≥ 1 Mb
Scaffold N50 length	44.26 Mb	= chromosome N50
Consensus quality (QV)	Primary: 50.9; alternate: 53.1; combined: 52.2	≥ 40
k-mer completeness	Primary: 82.86%; alternate: 78.18%; combined: 98.50%	≥ 95%
BUSCO	C:96.8% [S:95.5%; D:1.3%]; F:1.5%; M:1.7%; n:3 640	S > 90%; D < 5%
Percentage of assembly assigned to chromosomes	95.16%	≥ 90%

Full text

Species taxonomy

Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Deuterostomia; Chordata; Craniata; Vertebrata; Gnathostomata; Teleostomi; Euteleostomi; Actinopterygii; Actinopteri; Neopterygii; Teleostei; Osteoglossocephalai; Clupeocephala; Euteleosteomorpha; Neoteleostei; Eurypterygia; Aulopa; Aulopiformes; Alepisauroidei; Bathysauridae; Bathysaurus; Bathysaurus mollis Günther, 1878 (NCBI:txid1126214)

Background

We present a chromosome-level genome sequence for Bathysaurus mollis Günther (1878), the highfin lizardfish. The assembly was produced using the Tree of Life pipeline from a specimen collected from the Porcupine Abyssal Plain Sustained Observatory in the Northeast Atlantic ( Figure 1). This assembly was generated as part of the Darwin Tree of Life Project, which aims to generate high-quality reference genomes for all named eukaryotic species in Britain and Ireland to support research, conservation, and the sustainable use of biodiversity ( Blaxter et al., 2022).

Photographs of the Bathysaurus mollis Günther, 1878 specimen (DISCOLL_JC231_082_023; fBatMol1) used for genome sequencing.Top: lateral view of whole specimen on board prior to tissue sampling and preservation. Bottom: Dorsal detail view of head of the same specimen.

There are two valid species of the genus Bathysaurus, B. ferox Günther, 1878 and B. mollis, both of which are bathydemersal species found in deep waters circumglobally at tropical and temperate latitudes. Bathysaurus ferox is known from depths of 1 000 to 2 500 m, while B. mollis is generally known from 2 500 to at least 4 850 m ( Sulak et al., 1985). Both species have been shown to be synchronous hermaphrodites and are predominantly piscivorous ( Sulak et al., 1985). There are currently no known threats to B. mollis and because of the depth distribution of this species, it is unlikely to be threatened by anthropogenic disturbances and is considered by the IUCN Red list to be of Least Concern ( de Bruyne et al., 2015).

The two species can be distinguished by their colouration ( Bathysaurus mollis is unpigmented with pale translucent flesh, while B. ferox is generally pigmented and darker grey-brown), by their dorsal fins ( B. mollis has the dorsal fin base shorter than its head, whereas in B. ferox the dorsal fin base is much longer than its head); and by the possession of an adipose fin in B. mollis (lacking in B. ferox) ( Sulak et al., 1985).

Another chromosome-level assembly for this species is also available (GCA_048564825.1; submitted by IDSSE) (NCBI datasets, O’Leary et al., 2024).

Methods

Sample acquisition and DNA barcoding

The specimen used for genome and RNA sequencing was an adult Bathysaurus mollis Günther, 1878. The sample was collected during RRS James Cook cruise, JC231, using an OTSB14 (semi-balloon otter trawl, 14 m headrope; Merritt & Marshall, 1981) from the Porcupine Abyssal Plain Sustained Observatory (PAP-SO) site located in the NE Atlantic in international waters (48° 53.176' N, 16° 27.503' W to 48° 53.151' N, 16° 36.704' W, at 4840–4844 m depth, on 13th May 2022). The PAP-SO site has been the focus of an open ocean and deep-seabed study programme since 1985 ( Hartman et al., 2021; Hartman, 2022). Once on board the trawl sample was washed in filtered sea water, and selected specimens were photographed and tissue samples removed prior to fixation in 95% ethanol or 4% buffered formaldehyde. Part of the dissected tissue sample was placed into 95% ethanol for barcoding, and the rest of the tissue was placed into a 0.7 ml cryovial and preserved without fixative at –85 °C for whole genome sequencing. The specimen was sampled by Chris Fletcher and was identified by Tammy Horton based on morphology. Voucher tissue material is lodged at the Natural History Museum in London (NHM registration number NHMUK014453704; ToLID fBatMol1), while the voucher specimen was initially preserved in 4% buffered formaldehyde and transferred to 80% ethanol and is retained in the Discovery Collections, National Oceanography Centre, Southampton with specimen number DISCOLL_JC231_082_023. For the Darwin Tree of Life sampling and metadata approach, refer to Lawniczak et al. (2022).

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 specimen and stored in ethanol, while the remaining parts were shipped on dry ice to the Wellcome Sanger Institute (WSI) (see the protocol). 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 are available on protocols.io.

Nucleic acid extraction

Protocols for high molecular weight (HMW) DNA extraction developed at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory are available on protocols.io ( Howard et al., 2025). The fBatMol1 sample was weighed and triaged to determine the appropriate extraction protocol. Muscle tissue was homogenised by powermashing using a PowerMasher II tissue disruptor. HMW DNA was extracted using the Automated MagAttract v2 protocol. DNA was sheared into an average fragment size of 12–20 kb following the Megaruptor®3 for LI PacBio protocol. Sheared DNA was purified by automated SPRI (solid-phase reversible immobilisation). 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. For this sample, the final post-shearing DNA had a Qubit concentration of 2.84 ng/μL and a yield of 369.20 ng, with a fragment size of  kb. The 260/280 spectrophotometric ratio was 2.98, and the 260/230 ratio was 6.41.

RNA was extracted from muscle tissue of fBatMol1 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol. 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.

PacBio HiFi library preparation and sequencing

Library preparation and sequencing were performed at the WSI Scientific Operations core. Libraries were prepared using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, California, USA), following the manufacturer’s instructions. The kit includes reagents for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead clean-up, and nuclease treatment. Size selection and clean-up were performed using diluted AMPure PB beads (Pacific Biosciences). DNA concentration was quantified using a Qubit Fluorometer v4.0 (ThermoFisher Scientific) and the Qubit 1X dsDNA HS assay kit. Final library fragment size was assessed with the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) using the gDNA 55 kb BAC analysis kit.

The sample was sequenced on a Revio instrument (Pacific Biosciences). The prepared library was normalised to 2 nM, and 15 μL was used for making complexes. Primers were annealed and polymerases bound to generate circularised complexes, following the manufacturer’s instructions. Complexes were purified using 1.2X SMRTbell beads, then diluted to the Revio loading concentration (200–300 pM) and spiked with a Revio sequencing internal control. The sample was sequenced on a Revio 25M SMRT cell. The SMRT Link software (Pacific Biosciences), a web-based workflow manager, was used to configure and monitor the run and to carry out primary and secondary data analysis.

Hi-C

Sample preparation and crosslinking *

The Hi-C sample was prepared from 20–50 mg of frozen muscle tissue of the fBatMol1 sample using the Arima-HiC v2 kit (Arima Genomics). Following the manufacturer’s instructions, tissue was fixed and DNA crosslinked using TC buffer to a final formaldehyde concentration of 2%. The tissue was homogenised using the Diagnocine Power Masher-II. Crosslinked DNA was digested with a restriction enzyme master mix, biotinylated, and ligated. Clean-up was performed with SPRISelect beads before library preparation. DNA concentration was measured with the Qubit Fluorometer (Thermo Fisher Scientific) and Qubit HS Assay Kit. The biotinylation percentage was estimated using the Arima-HiC v2 QC beads.

Hi-C library preparation and sequencing *

Biotinylated DNA constructs were fragmented using a Covaris E220 sonicator and size selected to 400–600 bp using SPRISelect beads. DNA was enriched with Arima-HiC v2 kit Enrichment beads. End repair, A-tailing, and adapter ligation were carried out with the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs), following a modified protocol where library preparation occurs while DNA remains bound to the Enrichment beads. Library amplification was performed using KAPA HiFi HotStart mix and a custom Unique Dual Index (UDI) barcode set (Integrated DNA Technologies). Depending on sample concentration and biotinylation percentage determined at the crosslinking stage, libraries were amplified with 10 to 16 PCR cycles. Post-PCR clean-up was performed with SPRISelect beads. Libraries were quantified using the AccuClear Ultra High Sensitivity dsDNA Standards Assay Kit (Biotium) and a FLUOstar Omega plate reader (BMG Labtech).

Prior to sequencing, libraries were normalised to 10 ng/μL. Normalised libraries were quantified again and equimolar and/or weighted 2.8 nM pools. Pool concentrations were checked using the Agilent 4200 TapeStation (Agilent) with High Sensitivity D500 reagents before sequencing. Sequencing was performed using paired-end 150 bp reads on the Illumina NovaSeq X.

RNA library preparation and sequencing

Libraries were prepared using the NEBNext ^®^ Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer’s instructions. Poly(A) mRNA in the total RNA solution was isolated using oligo(dT) beads, converted to cDNA, and uniquely indexed; 14 PCR cycles were performed. Libraries were size-selected to produce fragments between 100–300 bp. Libraries were quantified, normalised, pooled to a final concentration of 2.8 nM, and diluted to 150 pM for loading. Sequencing was carried out on the Illumina NovaSeq X to generate 150-bp paired-end reads.

Genome assembly

Prior to assembly of the PacBio HiFi reads, a database of k-mer counts ( k = 31) was generated from the filtered reads using FastK. GenomeScope2 ( Ranallo-Benavidez et al., 2020) was used to analyse the k-mer frequency distributions, providing estimates of genome size, heterozygosity, and repeat content.

The HiFi reads were assembled using Hifiasm ( Cheng et al., 2021) with the --primary option. The Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019), and the contigs were scaffolded in YaHS ( Zhou et al., 2023) with the --break option for handling potential misassemblies. 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. TreeVal was used to generate the flat files and maps for use in curation. Manual curation was conducted primarily in PretextView and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Manual corrections included 53 breaks and 150 joins. This reduced the scaffold count by 6.6% and increased scaffold N50 by 1.7%. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation. PretextSnapshot was used to generate a Hi-C contact map of the final assembly.

Assembly quality assessment

The Merqury.FK tool ( Rhie et al., 2020) was run in a Singularity container ( Kurtzer et al., 2017) to evaluate k-mer completeness and assembly quality for the primary and alternate haplotypes using the k-mer databases ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.

The genome was analysed using the BlobToolKit pipeline, a Nextflow implementation of the earlier Snakemake version ( Challis et al., 2020). The pipeline aligns PacBio reads using minimap2 ( Li, 2018) and SAMtools ( Danecek et al., 2021) to generate coverage tracks. It runs BUSCO ( Manni et al., 2021) using lineages identified from the NCBI Taxonomy ( Schoch et al., 2020). For the three domain-level lineages, BUSCO genes are aligned to the UniProt Reference Proteomes database ( Bateman et al., 2023) using DIAMOND blastp ( Buchfink et al., 2021). The genome is divided into chunks based on the density of BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database with DIAMOND blastx. Sequences without hits are chunked using seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). The BlobToolKit suite consolidates all outputs into a blobdir for visualisation. The BlobToolKit pipeline was developed using nf-core tooling ( Ewels et al., 2020) and MultiQC ( Ewels et al., 2016), with containerisation through Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017).

Genome sequence report

Sequence data

PacBio sequencing of the Bathysaurus mollis specimen generated 27.55 Gb (gigabases) from 2.56 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 796.29 Mb, with a heterozygosity of 1.61% and repeat content of 31.45% ( Figure 2). These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 32× coverage. Hi-C sequencing produced 88.96 Gb from 589.11 million reads, which were used to scaffold the assembly. RNA sequencing data were also generated and are available in public sequence repositories. Table 1 summarises the specimen and sequencing details.

Frequency distribution of k-mers generated using GenomeScope2.The plot shows observed and modelled k-mer spectra, providing estimates of genome size, heterozygosity, and repeat content based on unassembled sequencing reads.

Assembly statistics

The primary haplotype was assembled, and contigs corresponding to an alternate haplotype were also deposited in INSDC databases. The final assembly has a total length of 1 065.77 Mb in 1 095 scaffolds, with 4 638 gaps, and a scaffold N50 of 44.26 Mb ( Table 2).

Most of the assembly sequence (95.16%) was assigned to 24 chromosomal-level scaffolds. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 3; Table 3).

Hi-C contact map of the Bathysaurus mollis genome assembly.Assembled chromosomes are shown in order of size and labelled along the axes, with a megabase scale shown below. The plot was generated using PretextSnapshot.

Table 3.: Chromosomal pseudomolecules in the primary genome assembly of Bathysaurus mollis fBatMol1.

The mitochondrial genome was also assembled (length 16.68 kb, OZ257237.1). This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.

The combined primary and alternate assemblies achieve an estimated QV of 52.2. The k-mer completeness is 82.86% for the primary assembly, 78.18% for the alternate haplotype, and 98.50% for the combined assemblies ( Figure 4).

Evaluation of k-mer completeness using MerquryFK.This plot illustrates the recovery of k-mers from the original read data in the final assemblies. The horizontal axis represents k-mer multiplicity, and the vertical axis shows the number of k-mers. The black curve represents k-mers that appear in the reads but are not assembled. The green curve corresponds to k-mers shared by both haplotypes, and the red and blue curves show k-mers found only in one of the haplotypes.

BUSCO v.5.7.1 analysis using the actinopterygii_odb10 reference set ( n = 3 640) identified 96.8% of the expected gene set (single = 95.5%, duplicated = 1.3%). The snail plot in Figure 5 summarises the scaffold length distribution and other assembly statistics for the primary assembly. The blob plot in Figure 6 shows the distribution of scaffolds by GC proportion and coverage.

Assembly metrics for fBatMol1.1.The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1 000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the actinopterygii_odb10 set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer.

BlobToolKit GC-coverage plot for fBatMol1.1.Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available on the BlobToolKit viewer.

Table 4 lists the assembly metric benchmarks adapted from Rhie et al. (2021) and the Earth BioGenome Project Report on Assembly Standards September 2024. The EBP metric, calculated for the primary assembly, is 5.C.Q50.

Table 4.: Earth Biogenome Project summary metrics for the Bathysaurus mollis assembly.

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. 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 materialLegality 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.