Abstract
The fourfinger threadfin (Eleutheronema tetradactylum) is a euryhaline fish distributed across the Indo-West Pacific, from the Persian Gulf to Australia. However, the lack of high-quality genomic resources has limited genomic-level studies of its evolution, conservation, and aquaculture. Here, we present the first Near telomere-to-telomere (T2T) genome assembly of E. tetradactylum, generated using Pacific Biosciences (PacBio) High-Fidelity (HiFi) sequencing, Oxford Nanopore Technologies (ONT) ultra-long reads, and Hi-C chromatin conformation capture. The final assembly spans 585.38 Mb across 83 contigs, with a contig N50 of 22.14 Mb and 98.76% of sequences anchored to 26 chromosomes. We annotated 22,362 protein-coding genes and identified that repetitive sequences constitute 18.09% of the genome. This high-quality T2T assembly demonstrates significant improvements in contiguity and completeness over previously available genomes, providing an invaluable resource to accelerate genetic research, advance molecular breeding, and inform conservation strategies for E. tetradactylum.
Similar content being viewed by others
Background & Summary
The fourfinger threadfin (Eleutheronema tetradactylum), a member of the family Polynemidae1, is a euryhaline pelagic fish inhabiting offshore waters of the Indo-West Pacific, spanning from the Persian Gulf to Australia2,3. Distinguished by its elongated, flattened body and four filamentous pectoral fins (Fig. 1), this species employs these fins for sensory detection of prey. As a commercially valuable species, E. tetradactylum is highly regarded in aquaculture for its rapid growth rate and high-quality meat4. However, overfishing and habitat degradation have led to significant population declines, resulting in its classification as Near Threatened on the International Union for Conservation of Nature (IUCN) Red List in 20145. Despite its importance, research has been constrained by a lack of high-quality genomic data, although studies have explored its population structure6,7, disease control8,9, farming techniques10, reproduction, basic biology11,12, and responses to environmental stressors2,13.
The map of Eleutheronema tetradactylum.
Although chromosome-level genome assemblies of E. tetradactylum have been published14, they are constrained by gaps, structural discontinuities, and incomplete annotations. Recent advancements in long-read sequencing technologies, such as PacBio and ONT, combined with improved assembly algorithms, have enabled the production of gap-free telomere-to-telomere (T2T) genomes. These assemblies resolve fragmented regions, enhance chromosomal continuity, and facilitate comprehensive variant detection15,16.
In this study, we generated a Near T2T genome assembly for E. tetradactylum using PacBio HiFi reads, ONT ultra-long reads, and Hi-C data. The assembly totals 585.38 Mb, comprising 83 contigs with an N50 of 22.14 Mb, and anchors 98.76% of sequences to 26 chromosomes. We annotated 22,362 protein-coding genes, achieving BUSCO completeness scores of 99.53% for the genome and 99.04% for annotations. This resource surpasses prior assemblies in contiguity and completeness, providing a foundational tool for advancing genetic, evolutionary, and conservation research on E. tetradactylum.
Methods
Sample collection
A two-year-old male E. tetradactylum, sourced from a local fishery farm in Zhanjiang, Guangdong Province, China, was used for this study. Tissues including muscle, eye, brain, liver, heart, spleen, kidney, and gill, were collected for genomic and transcriptomic sequencing. All samples were flash-frozen in liquid nitrogen and stored at −80 °C. was All procedures were approved by the Institutional Review Board on Bioethics and Biosafety of BGI-Shenzhen, China (No. FT18134).
Library construction and sequencing
Genomic DNA was extracted from muscle tissue for PacBio Single Molecule Real-Time (SMRT), Hi-C, Oxford Nanopore Technologies (ONT), and short-read genome survey sequencing. For SMRT sequencing, high-quality DNA was used to construct genomic libraries according to PacBio’s standard protocol (Pacific Biosciences, CA, USA) and sequenced on the PacBio Sequel II platform (Pacific Biosciences, Menlo Park, CA, USA) in Circular Consensus Sequence (CCS) mode. The raw data was filtered to obtain high-precision HiFi reads.
For ONT sequencing, an ultra-long library was constructed and sequenced on a PromethION flow cell (Oxford Nanopore Technologies Co., UK). Raw reads were filtered for quality (QV ≥ 7) using base-calling software, adapters were removed with Porechop (https://github.com/rrwick/Porechop), and reads shorter than 30 kb or with mean quality <90% were discarded using Filtlong (https://github.com/rrwick/Filtlong).
Hi-C libraries were constructed from muscle tissue following established protocols17. Tissue was cross-linked with formaldehyde, digested with a restriction enzyme, biotin-labeled, and ligated. After reversing cross-links and purifying DNA, fragments were sheared to ~300 bp, and paired-end libraries were sequenced on the DNBSEQ platform.
Total RNA was extracted from eight tissues (eye, brain, liver, heart, spleen, kidney, muscle, gill) using TRIzol reagent (Invitrogen). Paired-end sequencing was performed on the MGI-SEQ. 2000 platform.
Sequencing generated 32.77 Gb of HiFi data, 31.74 Gb of ONT data, 139.39 Gb of Hi-C data, and 21.94 Gb of RNA-seq data (Table 1).
Genome survey and assembly
For the genome survey, DNA libraries with 300–400 bp inserts were constructed. Then, DNA was purified, quantified, and sequenced from both ends using the DNBSEQ platform to obtain raw reads. Quality filtering of raw reads was performed using Fastp (v0.23.2; parameters: default)18, and K-mer frequency (K = 21) was calculated with Jellyfish (v2.3.0; parameters: -m 21 -s 1000000000)19. Based on K-mer distribution, GenomeScope 2.0 (v2.0; parameters: -k 21 -p 2)20 estimated the genome size to be 543.84 Mb, with a peak 21-mer depth of 120 (Fig. 2). The heterozygosity and repeat rates were found to be 0.545% and 10.328%, respectively. Smudgeplot (v0.2.3dev; parameters: -k21 -m100 -ci1 -cs1000)20 determined the species’ ploidy as AB type, indicating diploidy (Table 2).
Overview of the 21-mer frequency distribution in the E. tetradactylum genome. The x-axis indicates the coverage of the K-mer, the y-axis represents the k-mer frequency for a given depth.
The draft assembly of E. tetradactylum was performed using HiFi data combined with ONT ultra-long reads and Hi-C reads. The assembly was carried out with HiFiasm (v0.19.6; parameters: default)21, followed by redundancy removal with Purge Haplotigs (v1.0.4; parameters: default)22. This high-quality genome assembly served as the foundation for subsequent construction of chromosomes using the Hi-C reads.
Hi-C reads were aligned to the draft23, and the 3D-DNA pipeline, which included splitting, anchoring, sorting, orienting, and merging contigs or scaffolds, was employed to achieve chromosome-level scaffolding24. An interaction matrix was generated with Juicer (v1.5; parameters: chr_num 24)25 and manually refined using Juicebox (v1.11.08; parameters: default)26.
Ultra-long Oxford Nanopore Technologies (ONT) reads were aligned to chromosomes using minimap2 (v2-2.24; parameters: ont: -ax map-ont ccs: -ax map-hifi)27 to generate consensus sequences. These consensus sequences were then aligned to the ends of the chromosomes using blastn (v2.11.0+; parameters: -outfmt 7), and sequences with coverage ≥90% were used to replace the telomere sequences on the chromosomes based on their alignment positions. Gaps between contigs were filled using TGS-GapCloser (v1.2.0; parameters:–min_nread 10)28 by leveraging the coverage information between ultra-long ONT reads and the assembled contigs to perform contig extension. Subsequent polishing was carried out with Pilon (v1.23; parameters:–fix all–changes)29 using short-read sequencing data to correct errors in the extended and gap-filled genome, yielding the final telomere-to-telomere assembly of E. tetradactylum.
The final assembly spans 585.38 Mb across 77 scaffolds (26 chromosomes), with scaffold N50 of 23.88 Mb, contig N50 of 22.14 Mb, and 98.76% anchoring rate (Table 3). A Hi-C interaction heatmap confirmed high-quality chromosome assignments (Fig. 3). A total of 36 telomeric sequences were identified at the ends of the 26 chromosomes by searching the entire genome for the telomeric repeat motif (TTAGGG) (Table 4). The genomic positions of these telomeres and their distribution across contigs were annotated and visualized (Fig. 4).
Hi-C interaction heatmap of the E. tetradactylum genome. The x- and y-axes represent genomic positions (N*bin). Color intensity (yellow: low; red: high) indicates interaction strength. The first 26 squares represent the 26 chromosomes, followed by unanchored sequences.
An overview of the T2T gap-free reference genome of E. tetradactylum.
Repeats annotation
Repetitive elements were identified using a combination of de novo and homology-based approaches. Tandem repeats were predicted with TRF (v4.09; default)30. Homology searches employed RepeatMasker (v4.0.9; default)31 against the RepBase library (http://www.girin-st.org/repbase). Additionally, RepeatModeler (open-4.0.9; parameters: default)32 and LTR_FINDER_parallel (v1.0.7; parameters: default)33 were used to construct a de novo repeat library for E. tetradactylum, followed by a further de novo prediction using RepeatModeler. By integrating results from TRF, RepeatMasker, RepeatProteinMask, and de novo methods, and subsequently removing redundancies, we determined that repeat sequences and transposable elements (TEs) constitute approximately 18.09% and 16.69% of the E. tetradactylum genome, respectively (Fig. 5). Of which, repetitive DNAs, LINEs, SINEs and LTRs covered 8.69%, 3.19%, 0.29% and 1.70% of the entire genome, respectively (Table 5). This repeat content is comparable to that in Lates calcarifer (18.6%)34 but higher than in oreochromis niloticus (14%)35.
Genomic landscape of the E. tetradactylum chromosome-level assembly. Metrics were calculated using a window size of approximately 200 kb. Circos plot from the outer to the inner layers represents the following: (a) GC content (range: 36%–56%); (b) gene density (range: 0%–100%); (c) repeat density (range: 0%–100%); (d) LTR retroelement density (range: 0%–24%); (e) LINE density (range: 0%–61%); and (f) DNA transposon density (range: 0%–88%).
Gene prediction and functional annotation
To annotate genes in the E. tetradactylum genome, we conducted both structural and functional annotation. Gene structure annotation aimed to predict gene positions and structures through homology-based and de novo approaches, while functional annotation determined the biological roles and metabolic pathways associated with predicted gene products.
For gene structure annotation, we combined three strategies, including homology-based predictions, de novo prediction and RNA-sequencing-assisted prediction. we utilized Exonerate (v2.2.0; parameters: model protein2genome)36 and Liftoff (v1.6.3; parameters: showtargetgff 1)37 to align E. tetradactylum genome sequence with protein sequences from closely related species (Paralichthys olivaceus, Oryzias latipes, Carassius gibelio, Danio rerio, and Oryzias latipes) for homology-based prediction. De novo predictions were performed using AUGUSTUS (v3.3.2; parameters: default)38 and Genscan (v1.0; parameters: default)39. Additionally, RNA-seq data were mapped onto the E. tetradactylum genome, with transcripts and protein-coding genes predicted separately using StringTie (v1.3.5; parameters: default)40 and TransDecoder (v5.5.0; https://github.com/TransDecoder/TransDecoder) with default parameters. The predictions from these methods were integrated into a high-quality, non-redundant gene set using MAKER 2 (v2.31.10; parameters: default)41.
For gene function annotation, we compared the protein sequences of the genome derived from structure annotation against various databases, including GO42, KEGG43, Swissprot44, TrEMBL45, NR46, KOG (https://ftp.ncbi.nih.gov/pub/COG/KOG/) and AnimalTFDB47. This analysis, conducted using diamond (v2.0.14; parameters:–evalue 1e-05)48 software, provided insight into protein functions, metabolic pathways, and additional characteristics. To further identify conserved sequences, motifs, and structural domains, we analyzed Pfam49 and InterPro50 databases by using InterProScan (v5.61–93.0; parameters:–seqtype p–formats TSV–gote rms –pathways -dp)51. Pathway annotation was performed using KOBAS (v3.0; parameters: -t blastout: tab-sko)52 against the KEGG database Table 5.
Overall, we predicted 22362 protein-coding genes, with average gene length of 14620.72 bp, CDS length of 1811.74 bp,10.80 exons per gene, and exon length of 270.42 bp (Table 6). And then we predicted a total of 22,046 genes (98.59% of the total predicted genes) and 37,591 mRNA (98.71%) of the total predicted transcript) were successfully annotated (Fig. 6 and Table 7).
UpSet plot of gene functional annotations across nine databases: NR, SwissProt, TrEMBL, KOG, InterPro, GO, KEGG-ALL, KEGG-KO, and Pfam.
Non-coding RNAs were predicted using BLASTN(v2.11.0+; parameters: -evalue 1e-5)53 for rRNAs, tRNAscan-SE (v1.3.1; parameters: default)54 for tRNAs, and Infernal (v1.3.3) against Rfam (v14.8; parameters: cmscan --rfam --nohmmonly)55 for miRNAs and snRNAs. We identified 791 miRNAs, 1594 tRNAs, 1102 rRNAs, and 651 snRNA (Table 8).
Genome collinearity analysis
To investigate the conservation of genome structure, a synteny analysis was performed between the coding genes of E. tetradactylum and a related species, E. rhadinum, using JCVI (v1.1.22; parameters: “jcvi.compara.catalog ortholog --dbtype = prot --cscore 0.99 jcvi.compara.synteny screen --minspan = 70–align-chromosomes”)56. Both species share a 2n = 52 karyotype and exhibit high collinearity, indicating conserved synteny (Fig. 7).
Synteny analysis between E. tetradactylum and E. rhadinum genomes.
Data Records
The final telomere-to-telomere genome assembly for E. tetradactylum have been deposited in the National Center for Biotechnology Information (NCBI) GenBank database under accession number JBIEKL00000000057. Annotated coding sequences and protein sequences have been submitted to Figshare (https://doi.org/10.6084/m9.figshare.30164734)58. Raw sequencing reads (HiFi, Hi-C, ONT, genome survey, and RNA-seq) are deposited in the NCBI Sequence Read Archive (SRA) under accession number SRP53881059. All data are publicly accessible without restriction.
Technical Validation
Genome assembly and gene annotation quality assessment
Assembly and annotation completeness were evaluated with BUSCO (v5. 4. 3; parameters: default)60 against the actinopterygii_odb10 lineage. The genome recovered 99.53% BUSCOs (99.29% single-copy, 0.25% duplicated, 0.44% fragmented, 0.03% missing; Table 9). Annotations recovered 99.04% (84.09% single-copy, 14.95% duplicated, 0.33% fragmented, 0.63% missing; Table 9).
The PacBio HiFi reads were aligned to the assembly using minimap2 (v2.12, parameters: -ax map-pb)27, achieving 99.72% mapping and 99.84% coverage. GC content and depth were uniform across 100-kb windows (Fig. 8). Short reads were aligned with samtools (v1.17, parameters: sort -m 1 G)27, picard (v2.25.6; https://broadinstitute.github.io/picard/) and, GATK(v4.4.0.0; https://broadinstitute.github.io/picard/), revealing heterozygous SNP and InDel rates of 0.279% and 0.111%, with no homozygosity.
GC content and sequencing depth distribution. The x-axis represents the GC content; the y-axis represents the average depth.
In this study, we successfully achieved a T2T assembly for ten chromosomes: Chr1, Chr4, Chr5, Chr6, Chr7, Chr9, Chr10, Chr11, Chr12, and Chr26. For the remaining chromosomes, telomeres were identified at only one terminus. The difficulty in achieving complete T2T status for these sequences is likely attributable to the presence of recalcitrant genomic regions characterized by high complexity and extreme repetitive content (Fig. 5). Despite the utilization of current ONT ultra-long reads, the structural intricacy of these regions remains challenging to fully resolve. We anticipate that future advancements in sequencing read lengths and the continuous refinement of T2T assembly algorithms will eventually overcome these limitations, enabling the complete, gap-free assembly of the entire E. tetradactylum genome.
Data availability
The final telomere-to-telomere genome assembly for E. tetradactylum is available under GenBank accession JBIEKL00000000057, and comprehensive annotation files including structural annotations in GFF3 format and genomic sequences in FASTA format are provided via Figshare (https://doi.org/10.6084/m9.figshare.30164734)58. All raw sequencing data (HiFi, Hi-C, ONT, genome survey, and RNA-seq) generated in this study are available from NCBI Sequence Read Archive (SRA) under accession number SRP53881059.
Code availability
No custom code was developed for this study. All genome assembly, annotation, and validation analyses were performed using publicly available bioinformatics software with standard protocols and default parameters, as described in the Methods section.
References
Motomura, H. Threadfins of the world (Family Polynemidae): An annotated and illustrated catalogue of polynemid species known to date. (Food and Agricultural Organization, https://api.semanticscholar.org/CorpusID:128056442, 2004).
Ma, X. & Wang, W. X. Unveiling Osmoregulation and Immunological Adaptations in Eleutheronema tetradactylum Gills through High-Throughput Single-Cell Transcriptome Sequencing. Fish & shellfish immunology. 154, 109878, https://doi.org/10.1016/j.fsi.2024.109878 (2024).
Motomura, H., Iwatsuki, Y., Kimura, S. & Yoshino, T. Revision of the Indo-West Pacific polynemid fish genus Eleutheronema (Teleostei: Perciformes). Ichthyological Research. 49, 47–61, https://doi.org/10.1007/s102280200005 (2002).
Huang, C. T. et al. Bioeconomic evaluation of Eleutheronema tetradactylum farming: A case study in Taiwan. Fisheries Science. 88, 437–447, https://doi.org/10.1007/s12562-022-01591-4 (2022).
IUCN. The IUCN Red List of Threatened Species. Version 2015-4. Available at: https://www.iucnredlist.org/species/46087646/57168342#bibliography (2015).
Moore, B. et al. Stock structure of blue threadfin Eleutheronema tetradactylum across northern Australia, as indicated by parasites. Fish Biology. 78, 923–936, https://doi.org/10.1111/j.1095-8649.2011.02917.x (2011).
Xuan, Z. & Wang, W. X. Diversity of life history and population connectivity of threadfin fish Eleutheronema tetradactylum along the coastal waters of Southern China. Scientific Reports. 13, 3976, https://doi.org/10.1038/s41598-023-31174-x (2023).
Azad, I. S., Al-Yaqout, A., El-Dakour, S., Kawahara, S. & Al-Roumi, M. First record of iridovirus (ISKNV) infections in Fourfinger threadfin from Kuwait. Journal of King Saud University – Science. 36, 103393–103393, https://doi.org/10.1016/j.jksus.2024.103393 (2024).
Bharadhirajan, P. et al. Prevalence of copepod parasite (Lernaeenicus polynemi) infestation on Eleutheronema tetradactylum from Pazhayar coastal waters, southeast coast of India. Journal of Coastal Life Medicine. 1, 258–261, https://doi.org/10.12980/JCLM.1.20133D154 (2013).
Abu Hena, M. K., Idris, M. H., Wong, S. K. & Kibria, M. M. Growth and survival of Indian salmon Eleutheronema tetradactylum (Shaw, 1804) in brackish water pond. Journal of Fisheries and Aquatic Science. 6, 479–484, https://doi.org/10.3923/jfas.2011.479.484 (2011).
Iqbal, T. H. et al. Feeding habits of four-finger threadfin fish, Eleutheronema tetradactylum, and its diet interaction with co-existing fish species in the coastal waters of Thailand. Peer J 11, e14688, https://doi.org/10.7717/peerj.14688 (2023).
Soe, K. K. et al. Reproductive characteristics of the hermaphroditic four-finger threadfin, Eleutheronema tetradactylum (Shaw, 1804), in tropical coastal waters. BMC zoology 8, 22, https://doi.org/10.1186/s40850-023-00181-w (2023).
Jin, J. H., Amenyogbe, E., Yang, Y., Wang, Z. L. & Lu, Y. Effects of ammonia nitrogen stress on the physiological, biochemical, and metabolic levels of the gill tissue of juvenile four-finger threadfin (Eleutheronema tetradactylum). Aquatic toxicology (Amsterdam, Netherlands). 274, 107049, https://doi.org/10.1016/j.aquatox.2024.107049 (2024).
Xiao, J., Tsim, K. W. K., Hajisamae, S. & Wang, W. X. Chromosome-level genome and population genomics provide novel insights into adaptive divergence in allopatric Eleutheronema tetradactylum. International Journal of Biological Macromolecules. 244, 125299, https://doi.org/10.1016/j.ijbiomac.2023.125299 (2023).
Deng, Y. et al. A telomere-to-telomere gap-free reference genome of watermelon and its mutation library provide important resources for gene discovery and breeding. Molecular Plant. 15, 1268–1284, https://doi.org/10.1016/j.molp.2022.06.010 (2022).
Zhang, Y. H. et al. The telomere-to-telomere genome of Fragaria vesca reveals the genomic evolution of Fragaria and the origin of cultivated octoploid strawberry. Hortic Research. 10, uhad027, https://doi.org/10.1093/hr/uhad027 (2023).
Belton, J. M. et al. Hi–C: A comprehensive technique to capture the conformation of genomes. Methods. 58, 268–276, https://doi.org/10.1016/j.ymeth.2012.05.001 (2012).
Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 34, i884–i890, https://doi.org/10.1093/bioinformatics/bty560 (2018).
Marçais, G. & Kingsford, C. A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics. 27, 764–770, https://doi.org/10.1093/bioinformatics/btr011 (2011).
Ranallo-Benavidez, T. R., Jaron, K. S. & Schatz, M. C. GenomeScope 2.0 and Smudgeplot for reference-free profiling of polyploid genomes. Nature Communications. 11, 1432, https://doi.org/10.1038/s41467-020-14998-3 (2020).
Cheng, H.-Y., Concepcion, G. T., Feng, X., Zhang, H. & Li, H. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nature Methods 18, 170–175, https://doi.org/10.1038/s41592-020-01056-5 (2021).
Roach, M. J., Schmidt, S. A. & Borneman, A. R. Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies. BMC bioinformatics. 19, 1–10, https://doi.org/10.1186/s12859-018-2485-7 (2018).
Burton, J. N. et al. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nature Biological. 31, 1119–1125, https://doi.org/10.1038/nbt.2727 (2013).
Kajitani, R. et al. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Research. 24, 1384–1395, https://doi.org/10.1101/gr.170720.113 (2014).
Durand, N. C. et al. Juicer provides a one-click system for analyzing loop-resolution Hi-C experiments. Cell Systems. 3, 95–98, https://doi.org/10.1016/j.cels.2016.07.002 (2016).
Durand, N. C. et al. Juicebox provides a visualization system for Hi-C contact maps with unlimited zoom. Cell Systems. 3, 99–101, https://doi.org/10.1016/j.cels.2015.07.012 (2016).
Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 34, 3094–3100, https://doi.org/10.1093/bioinformatics/bty191 (2018).
Xu, M. Y. et al. TGS-GapCloser: A fast and accurate gap closer for large genomes with low coverage of error-prone long reads. Gigascience. 9, giaa094, https://doi.org/10.1093/gigascience/giaa094 (2020).
Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLOS One. 9, e112963, https://doi.org/10.1371/journal.pone.0112963 (2014).
Benson, G. J. N. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Research. 27, 573–580, https://doi.org/10.1093/nar/27.2.573 (1999).
Tarailo‐Graovac, M. & Chen, N. S. Using RepeatMasker to identify repetitive elements in genomic sequences. Current Protocols in Bioinformatics. 25, 4. 10. 1-4. 10. 14, https://doi.org/10.1002/0471250953.bi0410s25 (2009).
Price, A. L., Jones, N. C. & Pevzner, P. A. De novo identification of repeat families in large genomes. Bioinformatics. 21, i351–i358, https://doi.org/10.1093/bioinformatics/bti1018 (2005).
Ou, S. & Jiang, N. LTR_FINDER_parallel: parallelization of LTR_FINDER enabling rapid identification of long terminal repeat retrotransposons. Mobile DNA. 10, 48, https://doi.org/10.1186/s13100-019-0193-0 (2019).
Vij, S. et al. Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding. PLOS Genetics. 12, e1005954, https://doi.org/10.1371/journal.pgen.1005954 (2016).
Shirak, A. et al. Identification of Repetitive Elements in the Genome of Oreochromis niloticus: Tilapia Repeat Masker. Mar Biotechnol 12, 121–125, https://doi.org/10.1007/s10126-009-9236-8 (2010).
Slater, G. S. C. & Birney, E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics. 6, 1–11, https://doi.org/10.1186/1471-2105-6-31 (2005).
Shumate, A. & Salzberg, S. L. Liftoff: accurate mapping of gene annotations. Bioinformatics. 37, 1639–1643, https://doi.org/10.1093/bioinformatics/btaa1016 (2021).
Stanke, M. et al. AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Research. 34, W435–W439, https://doi.org/10.1093/nar/gkl200 (2006).
Burge, C. & Karlin, S. Prediction of complete gene structures in human genomic DNA. Journal of Molecular Biology. 268, 78–94, https://doi.org/10.1006/jmbi.1997.0951 (1997).
Pertea, M. et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology. 33, 290–295, https://doi.org/10.1038/nbt.3122 (2015).
Holt, C. & Yandell, M. MAKER2: an annotation pipeline and genome-database management tool for second-generation genome projects. BMC Bioinformatics. 12, 1–14, https://doi.org/10.1186/1471-2105-12-491 (2011).
Ashburner, M. et al. Gene ontology: tool for the unification of biology. Nature Genetics. 25, 25–29, https://doi.org/10.1038/75556 (2000).
Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Research. 28, 27–30, https://doi.org/10.1093/nar/28.1.27 (2000).
Boeckmann, B. et al. The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Research. 31, 365–370, https://doi.org/10.1093/nar/gkg095 (2003).
Bairoch, A. & Apweiler, R. The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Research. 28, 45–48, https://doi.org/10.1093/nar/28.1.45 (2000).
Sayers, E. W. et al. Database resources of the national center for biotechnology information. Nucleic Acids Research. 50, D20–D26, https://doi.org/10.1093/nar/gkg033 (2022).
Shen, W.-K. et al. AnimalTFDB 4.0: a comprehensive animal transcription factor database updated with variation and expression annotations. Nucleic Acids Research. 51, D39–D45, https://doi.org/10.1093/nar/gkac907 (2023).
BuchfInk, B., Reuter, K. & Drost, H.-G. Sensitive protein a lignments at tree-of-life scale using DIAMOND. Nature methods. 18, 366–368, https://doi.org/10.1038/s41592-021-01101-x (2021).
Bateman, A. et al. The Pfam Protein Families Database. Nucleic Acids Research. 28, 263–266, https://doi.org/10.1093/nar/28.1.263 (2000).
Paysan-Lafosse, T. et al. InterPro in 2022. Nucleic Acids Research. 51, D418–D427, https://doi.org/10.1093/nar/gkac993 (2023).
Jones, P. et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. 30, 1236–1240, https://doi.org/10.1093/bioinformatics/btu031 (2014).
Bu, D. H. et al. KOBAS-i: intelligent prioritization and exploratory visualization of biological functions for gene enrichment analysis. Nucleic acids research. 49, W317–W325, https://doi.org/10.1093/nar/gkab447 (2021).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. Journal of Molecular Biology. 215, 403–410, https://doi.org/10.1016/S0022-2836(05)80360-2 (1990).
Lowe, T. M. & Eddy, S. R. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Research. 25, 955–964, https://doi.org/10.1093/nar/25.5.955 (1997).
Griffiths-Jones, S. et al. Rfam: annotating non-coding RNAs in complete genomes. Nucleic Acids Research. 33, D121–D124, https://doi.org/10.1093/nar/gki081 (2005).
Tang, H. et al. Synteny and Collinearity in Plant Genomes. Springer Netherlands. 320, 486–488, https://doi.org/10.1126/science.1153917 (2008).
Zhang, H. J. et al. A chromosome-level genome assembly of the four-finger threadfin (Eleut- heronema tetradactylum). GenBank https://identifiers.org/ncbi/insdc:JBIEKL000000000 (2024).
Zhang, H. J., et al. A telomere-to-telomere genome of the fourfinger threadfin (Eleutheronema tetradactylum). Figshare (https://doi.org/10.6084/m9.figshare.30164734).
NCBI Sequence Read Archive https://identifiers.org/ncbi/insdc.sra:SRP538810 (2024).
Manni, M., Berkeley, M. R., Seppey, M., Simão, F. A. & Zdobnov, E. M. BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Molecular biology and evolution. 38, 4647–4654, https://doi.org/10.1093/molbev/msab199 (2021).
Acknowledgements
The research was financially supported by the Guangdong Province Ordinary Colleges and Universities Key Field Special Project (Science and Technology Services for Rural Revitalization) (2023ZDZX4011), Guangdong Ocean University Aquaculture Excellent Young Talent Program (2024), and the Guangdong Province Ordinary Colleges and Universities Innovation Team Projects (2021KCXTD026; 2022KCXTD013).
Author information
Authors and Affiliations
Contributions
These authors contributed equally: Huijuan Zhang, Yifei Pan, Benxun Miao, Linjuan Wang, Minxuan Jin, Anna Zheng, Jiandong Zhang, Baogui Tang, Jiansheng Huang, Jing Li, Dee-hwa Chong and Zhongliang Wang. H.J.Z. and L.J.W. conceived the project. Y.F.P., B.X.M., M.X.J., A.N.Z., J.D.Z., B.G.T., J.S.H., J.L., D.C. and Z.L.W. collected the samples. H.J.Z., Y.F.P., B.X.M., M.X.J., A.N.Z., J.D.Z., B.G.T., J.S.H., J.L., D.C. and Z.L.W. performed the genome assembly, gene annotation and other bioinformatics analysis. H.J.Z. and L.J.W. wrote and revised the manuscript. B.G.T., J.S.H., J.L. and Z.L.W. revised the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Zhang, H., Pan, Y., Miao, B. et al. Near telomere-to-telomere genome assembly of the fourfinger threadfin (Eleutheronema tetradactylum). Sci Data 13, 39 (2026). https://doi.org/10.1038/s41597-025-06352-3
Received:
Accepted:
Published:
Version of record:
DOI: https://doi.org/10.1038/s41597-025-06352-3
