AbstractGlass catfish (Kryptopterus vitreolus) is commonly distributed in several Asian countries, such as Thailand, Malaysia, and Indonesia. It is renowned for its near-transparent appearance, which has drawn considerable attention for biomedical research and the tropical ornamental fish industry. Here, we successfully constructed the first telomere-to-telomere (T2T) chromosome-scale genome assembly for glass catfish, by integration of PacBio HiFi, Nanopore ONT ultra-long, and Hi-C sequencing technologies. The haplotypic assembly covers approximately 687.7 Mb in length, featuring a high contig N50 of 21.3 Mb. This assembly was then anchored into 32 chromosomes, presenting a complete set of 64 telomeres and 32 centromeres. It was predicted with 252.4 Mb of repetitive sequences and annotated with a total of 24,696 protein-coding genes. Subsequent BUSCO analysis revealed high genome completeness (up to 96.4%). This high-quality T2T genome assembly not only provides a valuable genetic resource for investigating the molecular mechanisms underlying transparency, but also supports in-depth studies on functional genomics, genetic diversity, and selective breeding for this economically important fish species.
Background & SummaryKryptopterus vitreolus (NCBI Taxonomy ID: 2012678), is a special species within the Siluridae family and the Siluriformes order. It is also named as “glass catfish” or “ghost catfish” due to its almost transparent appearance. It exhibits minimal pigmentation, displaying infrequent chromatic markings. Colors of black, metallic silver, and pale yellow are arranged linearly along its axis from cephalic to mid-body regions. Its intermuscular bones and internal organs can be directly seen through its muscle. The transparency gives it a strong adaptive edge, making this fish almost undetectable to predators. The glass catfish therefore lives smoothly in the complex river systems of some peninsular and southeastern areas of Thailand, Malaysia, and Indonesia1.Owing to its glass-like appearance, it holds particular appeal for aquatic enthusiasts and exhibition curators, and it is also a good model for probing pigmentation2,3, cell transplantation and isolation4, DNA immunization5 and vascular system studies6,7. Nevertheless, this fish has often been misidentified as either Kryptopterus minor or, more frequently, K. bicirrhis. Previous Ng & Kottelat’s study1 resolved this long-standing issue by classifying the glass catfish as a distinct species, named as K. vitreolus. Their report also updated Kryptopterus classification, noting the glass catfish’s clear and small build versus the more non-transparent and gigantic K. bicirrhis.In our previous glass catfish genome study8, we reported a draft chromosome-level genome assembly with identification of some key genes, such as tyrp1 and edn3, which play potential roles in pigmentation formation. However, this assembly contains too many gaps with low BUSCO values that led to a lot of missing fragments for shortcoming in genome completeness and details. Here, with large advancements in high-throughput sequencing technology, such as extra PacBio HiFi and Nanopore ONT (Oxford Nanopore Technologies) ultra-long sequencing techniques, we constructed a highly improved telomere-to-telomere (T2T) chromosome-scale genome assembly (with much higher scaffold N50 and better BUSCO) for glass catfish, which is complete in genome sequence with more details about telomeres and centromeres. This new genomic resource not only offers a high-quality assembly to work as a foundational genome reference for in-depth characterization and breeding of glass catfish, but also supports comparative and molecular studies on the regulation of pigmentation in various vertebrates.MethodsSample collection and DNA extractionThree glass catfish individuals (1.0 g) were obtained from a local aquarium fish market in Guangzhou, Guangdong, China. We extracted their genomic DNA (gDNA) from muscle tissue by using a TIANamp Genomic DNA Extraction Kit (Tiangen, Beijing, China) for subsequent whole genome sequencing9. A Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and a Nanodrop spectrophotometer (Thermo Fisher Scientific)10 were used respectively to measure gDNA concentration and purity, while gDNA integrity was verified on a 1.0% agarose gel.Library construction for genome and transcriptome sequencingFor PacBio HiFi long-read sequencing, DNA libraries were prepared by utilization of SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA), adhering meticulously to PacBio’s established protocols. Subsequently, these libraries were sequenced on a PacBio Sequel II platform11. High-accuracy consensus reads were generated using the Circular Consensus Sequencing (CCS, SMRT Link v11.0) software12, yielding 75.0 Gb of HiFi data (Table 1) with an N50 length of 19.9 kb.Table 1 Statistics of the T2T genome assembly and annotation (in this study) with comparison to the previous report.Full size tableFor ONT ultra-long sequencing, a library was prepared using an SQK-ULK001 kit according to the guidance from the manufacturer (Oxford Nanopore Technologies, UK). This library was then sequenced on a PromethION flow cell (Oxford Nanopore Technologies), and about 18.5 Gb of ultra-long reads (Table 1) with an N50 length of 56.2 kb were yielded. Subsequently, these data were processed by using NECAT v200221 with default parameters13.For high-throughput chromosome conformation capture (Hi-C) sequencing, we extracted gDNA, digested chromatin using the restriction enzyme MboI, and then conducted proximity ligation according to protocols outlined in previous studies14. In brief, gDNA was cross-linked, digested, biotin-labeled, ligated, and fragmented to 500 bp, followed by purification with streptavidin magnetic beads. Library quality and insert size were assessed by using a Qubit 3.0 Fluorometer and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. Libraries were sequenced on an Illumina NovaSeq. 6000 platform (Illumina, San Diego, CA, USA), generating ~101.7 Gb of 150 bp paired-end reads.For transcriptome sequencing, total RNA was isolated from muscle tissue by utilizing TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA integrity was assessed by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), and only those samples with an RNA Integrity Number (RIN) above 7.0 were proceeded for subsequent library preparation. Libraries were constructed employing the DNA nanoball (DNB) technology on a DNBSEQ T7 platform (MGI, BGI Shenzhen, China), and sequencing was performed on a MGISEQ-2000 platform (MGI) to generate 11.7 Gb of 150-bp paired-end reads.Genome assembly and quality evaluationA genome assembly of glass catfish was initially obtained by using NextDenovo15 (v2.2-beta.0, https://github.com/Nextomics/NextDenovo) with the following parameters: -rerun 3 -read_cutoff 1k -pa_correction 2, resulting in primary contigs with a total size of ~698.3 Mb and a contig N50 of 5.1 Mb. Subsequently, Hi-C sequencing data were aligned to these contigs using Bowtie2 v2.2.5 (configured with --very--sensitive -L 30 --score-min L, --0.6, --0.2–end-to-end –reorder)16, and then effective linkage information was identified by using HiC-Pro v2.8.117 under default settings, retaining only valid contact pairs to support anchoring of contigs into chromosomes.To orient, order, and cluster contigs into chromosome-level scaffolds, Juicer v1.5 (parameter: chr num 32)18 and 3D-DNA v170123 (parameters: -m haploid -r 2)19 were employed. Visualization and manual adjustments were facilitated to adjust mis-assemblies and eliminate redundant contigs by using Juicebox v1.11.0820. This primary chromosome-level genome assembly contained 421 gaps. To achieve a gap-free T2T chromosome-level assembly, LR_GapCloser v1.0 (parameters: -t 35 -m 1000000 -v 10000)21 and TGS-GapCloser v1.0.1 (parameter: -min_match 2000)22 were sequentially applied to fill these gaps. The final haplotypic genome assembly anchored 32 chromosomes (Figs. 1 and 2) with a total length of 687.7 Mb (Fig. 1, Table 2), which represents ~98.5% of the assembled contigs. The contig N50 value of these anchored chromosomes was increased to 21.3 Mb, which is much higher than our previous report8 (1.2 Mb; Table 1).Fig. 1A chromosomal heatmap derived from Hi-C data. A total of 32 chromosomes (blue boxes) were successfully anchored.Full size imageFig. 2A Circos image of the 32 anchored chromosomes within the assembled haplotypic genome. Data from the outside to the inside include the length and number of each chromosome, gene density distribution (bin = 0.1 Mb), GC content (bin = 0.1 Mb), repetitive sequence density (bin = 0.1 Mb), and a fish photo (in the center).Full size imageTable 2 Summary of the complete T2T chromosomes (chr) in the assembled genome.Full size tableTo evaluate genome completeness and accuracy, we utilized Benchmarking Universal Single-Copy Orthologs v5.2.2 (BUSCO)23 with the Actinopterygii odb10 dataset as the reference. Moreover, genome quality was assessed by the K-mer-based tool Merqury v1.3 (https://github.com/marbl/merqury)24 with a K-mer size of 17. This analysis compared K-mer frequency distributions between the genome assembly and whole-genome sequencing data based on HIFI data, producing good metrics for genome completeness and quality values (QV).Repeat element annotationIn this T2T genome assembly of glass catfish, repetitive elements (REs) were identified through integration of de novo and homology-based annotation methods. For the de novo annotation, RepeatModeler v2.0.125 and LTR-FINDER v1.0.626 were employed to construct a customized repeat library. Subsequently, RepeatMasker version 4.1.027 was utilized with default settings to annotate REs within the assembled genome, employing Repbase transposable element (TE) library version 21.028 as the reference. In the homology-based methodology, Tandem Repeats Finder v4.07 was employed with fine-tuned parameters (2 7 7 80 10 50 2000 -d -h)29 to discern tandem repeat sequences. Concurrently, RepeatMasker v4.1.027 and RepeatProteinMask v4.1.027 were utilized to annotate transposable elements (TEs) within the assembled genome, with default configurations. Centromeric and telomeric sequences were identified through application of the quarTeT software30.The final analysis revealed that approximately 252.4 Mb of the glass catfish genome consisted of repetitive sequences (Fig. 3), constituting 36.7% of the total genome length (Table 1). The quarTeT results indicate that this genome assembly possesses intact telomere and centromere sequences on corresponding chromosomes, including a complete set of 64 telomeres and 32 centromeres (see more details in Fig. 3).Fig. 3Localization of telomeres and centromeres and the distribution pattern of repetitive sequences in the assembled genome of glass catfish. Arrows represent the complete set of 64 telomeres at both ends of the 32 chromosomes. One centromer is localized within each chromosome. Repetitive sequences are marked with different colors (a ruler in the top right is provided as the density reference).Full size imageGene prediction and functional annotationTo develop a comprehensive gene set for the glass catfish genome, we employed an integrated approach that combines homology-based annotation and transcriptome-derived annotation to accurately predict protein-coding genes. For the homology-based annotation, protein sequences of five representative teleost species were downloaded from the NCBI database. These species included Danio rerio (zebrafish; GCF_000002035.6), Oryzias latipes (medaka; GCF_002234675.1), Takifugu rubripes (Japanese puffer; GCF_901000725.2), Tetraodon nigroviridis (green spotted puffer; GCA_000180735.1) and Gasterosteus aculeatus (stickleback; GCA_016920845.1). These protein sequences were aligned to the assembled glass catfish genome by utilizing tBLASTn31 with an e-value threshold of 10−5. After obtaining alignments, gene structure refinement was conducted by using GeneWise v2.2.032, employing the following parameters: –blast_eval 1e-5, –align_rate 0.5, and –extend_len 500.Approximately 11.7 Gb of transcriptomic data were assembled into unigenes using Trinity v2.5.133, and then gene structures were predicted using PASA v2.3.334. The outcomes derived from above two methodologies were subsequently combined via the MAKER software35 (version specifics: max_dna_length = 300000, minimum_contig_size = 500, prediction_flanking_region = 500, annotation_error_threshold = 1.0, split_hit_length = 30000, allow_single_exon_genes = True, minimum_single_exon_gene_length = 250). For subsequent functional annotation, predicted protein sequences were subjected to a comparative analysis against three public databases, SwissProt36, TrEMBL37 and KEGG38, by employing BLASTP with a stringent e-value threshold of <1e-5. Gene Ontology (GO)39 annotation was additionally ascertained through application of InterProScan40.Finally, a total of 24,696 protein-coding genes were annotated, with an average mRNA length of 15,135.6 bp. Each gene had an average of 9.7 exons with an average exon length of 188.4 bp, an average intron length of 1,483.2 bp, and an average coding sequence (CDS) length of 1,827.3 bp. Approximately 98.4% (24,307 genes) of the predicted genes were annotated with at least one database, highlighting the high completeness and reliability of the glass catfish gene annotation (Table 1).Data RecordsThe final genome assembly of glass catfish was publicly accessible in the NCBI database under accession number PRJNA116962941. The genomic data are stored in the NCBI GenBank with the entry number GCA_044706155.142. Annotated coding sequences and protein sequences have been submitted to Figshare (https://doi.org/10.6084/m9.figshare.28333385)43. Additionally, raw reads obtained by PacBio, ONT and Hi-C reads are available in the NCBI database under accession numbers SRR3224786044, SRR3224786145 and SRR3224785946.Technical ValidationThe PacBio HiFi reads, aligned to the genome assembly utilizing Minimap2, achieved an impeccable mapping rate of 100.0%, and the HIC reads also gained a 100% mapping rate. Completeness was further evaluated with BUSCO v.5.2.2, using the Actinopterygii database (3,640 single-copy orthologs; OrthoDB v.10) as the reference. In summary, 96.4% (3,510) of BUSCO genes were complete, with 95.1% (3,462) being identified as single-copy, 1.3% (48) as duplicated, and a mere 0.6% (21) as fragmented. Protein-level BUSCO assessment with the Actinopterygii dataset revealed 92.5% (3,367) completeness, with 90.8% (3,306) single-copy, 1.7% (61) duplicated, and 2.4% (88) fragmented orthologs, confirming a high-quality gene annotation. A Merqury QV score of 39.7 further validated the high quality and reliability of our constructed genome assembly.
Code availability
All scripts and pipelines used for the genome assembly and gene annotation followed the standard manuals and protocols of the applied bioinformatics software. No specific code was developed for this study.
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Chao Bian or Qiong Shi.Ethics declarations
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The authors declare no competing interests.
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Reprints and permissionsAbout this articleCite this articleBian, C., Li, D., Wang, Y. et al. A telomere-to-telomere chromosome-scale genome assembly of glass catfish (Kryptopterus vitreolus).
Sci Data 12, 483 (2025). https://doi.org/10.1038/s41597-025-04841-zDownload citationReceived: 10 February 2025Accepted: 14 March 2025Published: 23 March 2025DOI: https://doi.org/10.1038/s41597-025-04841-zShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard
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