A telomere-to-telomere gap-free genome assembly of the protandrous hermaphrodite Asian seabass (Lates calcarifer)

Abstract

As a protandrous hermaphroditic fish species with natural sex change from male to female, Asian seabass (Lates calcarifer) represents an attractive model for studying sequential hermaphroditism. In this study, we constructed the first telomere-to-telomere (T2T) gap-free genome assembly of Asian seabass, by integration of MGI short-read, PacBio HiFi long-read, ONT ultra-long and Hi-C sequencing technologies. The haplotypic 614.19 Mb genome sequences were successfully anchored onto 24 chromosomes, demonstrating exceptional contiguity with a contig N50 of 26.57 Mb. Comprehensive annotation revealed precise localization of telomeric repeats and centromeric regions across various chromosomes. Good results from Merqury (QV: 57.8), CRAQ (99.45%) and BUSCO (100%) indicate a high level of accuracy for the assembled genome. ONT ultra-long and PacBio HiFi sequencing data were aligned with the assembly using minimap2, resulting in a mapping rate over 98%. Repetitive elements accounted for 18.18% (111.64 Mb) of the entire genome, and a total of 25,093 protein-coding genes were annotated. This high-quality T2T genome assembly provides a valuable genetic resource for in-depth comparative genomics, population genetics, molecular breeding, and functional studies of this economically important marine species. This reference assembly also facilitates investigations into the detailed molecular mechanisms underlying its unique reproductive strategy of the protandrous hermaphrodite Asian seabass.

Background & Summary

Sex determination is a genetic or epigenetic process that initiates and regulates the developmental trajectory of sexual differentiation, whereas sex differentiation encompasses the cascade of morphological and physiological events through which a bi-potential gonad progressively develops into either a testis or an ovary, culminating in the establishment of species-specific secondary sexual characteristics¹. Compared with those highly conserved sex determination systems in various mammals and birds, fishes exhibit remarkable diversity in sex determination patterns. They present more diversified sex determination modes than higher vertebrates, such as genetic sex determination (GSD), environmental sex determination (ESD), and the coexistence of both^2,3. Notably, among diverse environmental cues, temperature emerges as the most influential exogenous factor to modulate sexual development in fishes. Numerous species across different taxa have been documented to own thermally sensitive sex determination, where incubation temperature during critical developmental windows can override genotypic sex determinants. Good examples include European seabass (Dicentrarchus labrax)⁴, tilapia (Nile tilapia and Oreochromis niloticus)⁵, and Atlantic halibut (Hippoglossus hippoglossus)^6,7. These fishes exhibit interesting characteristics of temperature-dependent sex determination, and their sex ratios can change significantly with variations in environmental temperature during their hatching period.

In addition to gonochorism (separate sexes), fishes also exhibit hermaphroditism as an important reproductive strategy. Approximately 2% of teleost fishes are hermaphroditic, distributed across 27 families within 7 orders⁸. Sex change is a biological process in which an organism transitions from its original sex to another through specific physiological mechanisms. Organisms capable of naturally undergoing sex change are referred to hermaphrodites, which are typically categorized into protandrous (male-to-female) and protogynous (female-to-male)⁹. Common examples in these fishes include groupers, black seabream, clownfish, and ricefield eel^10–13.

Asian seabass holds substantial cultural and economic values throughout the tropical Indo-West Pacific region, serving as both a key fishery resource and a commercially important aquaculture species¹⁴. As a protandrous hermaphroditic fish¹⁵, it usually first develops into a male at 3–4 years of age, and then approximately 90% of individuals undergoes natural sex change to female by age 6¹⁶. Despite its remarkable reproductive strategy, the genetic mechanisms underlying sex change in Asian seabass remain poorly understood, as is the case for most hermaphroditic species. Genomic resources, including DNA markers, high-resolution linkage maps, transcriptomes, reference genome sequences along with their comprehensive annotations, play a pivotal role in supporting aquaculture. These valuable genetic resources provide a solid foundation for diverse applications, enabling comprehensive genetic investigations to support development of sophisticated artificial breeding strategies. Ultimately, they contribute to the sustainable expansion and increased productivity of international aquaculture industry¹⁴. Given the economic value of Asian seabass and its remarkable natural sex change, construction of its high-quality genome assembly is absolutely essential.

In this study, we combined MGI short-read, PacBio HiFi long-read, ONT (Oxford Nanopore Technologies) ultra-long, and Hi-C sequencing data to generate a high-fidelity T2T genome assembly of Asian seabass. This assembly was rigorously assessed for quality, and its key genomic features were systematically characterized. In fact, this gap-free and complete reference assembly represents a substantial improvement over any previous assembly of this species¹⁷. It will not only facilitate population genetic research and evolutionary study, but also provide an important genetic resource for molecular breeding and investigating molecular mechanisms of sex change in this economically important fish.

Methods

Sample collection

A male Asian seabass (Fig. 1A) was collected from a local aquaculture facility of the South China Sea Fisheries Research Institute under Chinese Academy of Fishery Sciences, which is located in Guangzhou City, Guangdong Province, China. Muscle tissue was sampled for whole-genome sequencing, including MGI short read, PacBio HiFi long read, ONT (Oxford Nanopore Technologies) Ultra-long and Hi-C sequencing technologies. Additionally, seven distinct tissues (such as gill, brain, liver, muscle, eye, testis, and skin) were collected for transcriptome sequencing (Table 1). Upon dissection into small fragments, the tissue samples were washed with ice-cold PBS (pH 7.4) to eliminate blood residues and contaminants. After removing outside liquid by blotting, these samples were rapidly frozen in liquid nitrogen and subsequently maintained at −80 °C before use. For transcriptome sequencing, frozen specimens were shipped in dry ice containers to the sequencing company (BGI, Shenzhen, Guangdong, China).

Fig. 1.

Asian seabass and its whole-genome sequence distribution. (A) A morphological image of the sequenced Asian seabass. (B) A k-mer (21-mer) distribution curve for estimation of the genome size.

Table 1.

Sequencing data of the Asian seabass genome and transcriptomes.

Type	Library type	Raw data (Gb)	Clean data (Gb)	Read N50/ length (bp)	Coverage of the genome (×)
DNA	MGI	37.41	33.69	150	54
	PacBio HiFi	/	90.47	18,366*	141
	ONT Ultra-long	/	61.34	71,701*	95
	Hi-C	102.32	93.8	150	133.33
RNA	Eye	6.104	5.236	150	/
	Muscle	6.207	5.625	150	/
	Skin	6.332	5.769	150	/
	Liver	7.007	6.377	150	/
	Gill	6.868	6.263	150	/
	Brain	6.335	5.783	150	/
	Testis	9.195	8.395	150	/

*For the PacBio HiFi and ONT Utra-long sequencing, this number is N50 of reads; for others, it denotes read length.

DNA extraction and genome sequencing

Genomic DNA (gDNA) was extracted from muscle tissue using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocols¹⁸. Fragment size, purity, and quantification of the extracted gDNA were assessed via 0.75% agarose gel electrophoresis, an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively.

For the MGI short-read sequencing, gDNA was randomly fragmented using a MGIEasy Universal DNA Library Preparation Kit (MGI, Shenzhen, China) to construct a library with an insert-size of 350 bp. Sequencing was performed on a DNBSEQ-T7 platform (MGI), generating 37.4 Gb of raw 150-bp paired-end reads, and then filtered by fastp v0.12.6¹⁹ (parameter: -n 0 -f 5 -F 5 -t 5 -T 5) to remove adaptor sequences and low-quality reads. Finally, a total of 33.69 Gb of clean reads (Table 1) were obtained for further data error correction and genome-size estimation.

For the PacBio HiFi sequencing, approximately 10 μg of high-quality gDNA was applied to construct a SMRTbell library following the manufacturer’s standard protocol (SMRTbell Express Template Prep Kit 2.0; Pacific Biosciences, Menlo Park, CA, USA), which was then sequenced on a PacBio Sequel II System using the circular consensus sequencing (CCS) technology. A total of 90.47 Gb of HiFi reads with a N50 of 18,366 bp were obtained (Table 1) using the CCS v6.0.0²⁰ (Circular Consensus Sequencing) software with the optimized parameter “-min-passes 3”.

Two ultra-long read libraries were constructed using Oxford Nanopore Technologies (ONT) protocols, which were sequenced on a PromethION platform (Oxford Nanopore Technologies Co., Littlemore, Oxford, UK). Raw reads were initially processed to eliminate those with a quality value (QV) lower than 7 using the NanoFilt v2.8.0²¹ software. Finally, a total of 1.54 million clean reads were retained, accumulating a substantial base count of 61.32 Gb. The average read length was 39.69 kb, with an N50 length of 71.17 kb (Table 1).

For the high-throughput chromosome conformation capture (Hi-C) sequencing, one Hi-C library was generated using a GrandOmics Hi-C kit (GrandOmics, Wuhan, Hubei, China) following the manufacturer’s protocol. In brief, gDNA was first cross-linked using a 4% formaldehyde solution to stabilize chromatin structures. Subsequently, the DNA was digested with the restriction enzyme MboI to introduce specific cleavage sites. Those resulting DNA fragments were then labeled with biotin-14-dCTP, allowing for incorporation of a detectable marker. The labeled DNA fragments were ligated using T4 DNA ligase to facilitate subsequent enrichment steps. Following ligation, the DNA was further digested to yield fragments in the size range of 200 to 600 bp. The library was sequenced on a DNBSEQ-T7 platform (MGI, Shenzhen, China) using a 150-bp paired-end model. The Hi-C sequencing technology generated 102.32 Gb of raw data. Subsequently, fastp v0.12.6¹⁹ was applied to filter adaptor sequences and low-quality reads. Finally, 93.8 Gb of Hi-C clean data were retained (Table 1) for chromosome assembly.

RNA extraction and transcriptome sequencing (RNA-seq)

Total RNA was extracted from seven tissues separately according to a standard Trizol protocol (Invitrogen, Frederick, MD, USA), followed by purification with a Qiagen RNeasy Mini Kit (Qiagen, Germantown, MD, USA). RNA concentration and integrity were measured using a NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. Only those RNA samples with OD260/280 ≥ 1.8 and RNA integrity ≥ 7.0 were selected for transcriptome sequencing. RNA was used for construction of a cDNA library followed the manufacture’s guideline, which was then sequenced on a HiSeq X Ten platform (Illumina, San Diego, CA, USA). A total of 48.07 Gb of transcriptome raw data were generated (Table 1), which aided in annotation of protein-coding genes and prediction of gene structures.

Genome-size estimation and construction of a T2T genome assembly

To estimate the genome size of Asian seabass, we employed jellyfish (v2.2.10)²² to perform k-mer counting with k = 21, and the parameters were set as ‘-m 21 -s 10 G -C’. Subsequently, a generated histogram was utilized as an input file for GenomeScope v2.0²³ to estimate genetic characteristics. This approach provided a sequence-derived estimate of the Asian seabass genome characteristics prior to assembly. Our analysis results show that the genome size of Asian seabass is approximately 576.74 Mb, with an estimated heterozygosity of about 0.46% (Fig. 1B) and repetitive sequences accounting for 32.79 Mb (5.69%).

Primary contigs were initially generated by assembling PacBio HiFi and ONT data using Hifiasm v0.19.8²⁴ with default parameters. Then, purge_dups v1.2.5²⁵ was employed to remove haplotypic and heterozygous duplications from the de novo assembly, yielding a final assembly with a total length of 614.08 Mb.

Using the preliminary assembly as the reference, Hi-C clean reads were utilized to construct chromosomes for Asian seabass. First, the Hi-C reads were mapped to the assembled contigs using bowtie2 v2.2.5 (–very-sensitive -L 20–score-min L, −0.6, −0.2–end-to-end)²⁶. Subsequently, the HiC-Pro v2.8.1²⁷ pipeline was applied to detect ligation products, retaining only valid paired reads for downstream analysis. Based on these valid reads, the primary assembly was clustered, ordered, and oriented into chromosomes using the Juicer v1.5²⁸ and 3D-DNA v3.0²⁹ software with parameters -m haploid -r 2 -c 24. Juicebox v1.11.08³⁰ was employed to visualize before manually adjusting the candidate assemblies.

To fill the remaining gaps, those corrected ultra-long ONT reads were applied to generate a gap-free genome assembly using TGS-GapCloser v1.2.1³¹ with optimized parameter “–min_match 1000–min_nread 3” and LR_Gapcloser v1.0³² with the parameter “-t 35 -m 1000000 -v 500”. The final genome assembly spans 614.19 Mb, and it is anchored onto 24 chromosomes (Fig. 2), among them the longest and the shortest are 31.85 Mb and 14.85 Mb, respectively (Table 2).

Fig. 2.

The first T2T genome assembly of Asian seabass. (A) Genome-wide chromatin interactions at a 500-kb resolution. Color blocks represent corresponding interactions, with various strengths from yellow (low) to red (high). (B) A Circos plot of the main genome features. From outside to inside include the 24 chromosomes, gene density, GC content, repetitive sequences density, and a colinear relationship among chromosomes of the Asian seabass genome assembly. Note that the density calculation window is set as 100 kb.

Table 2.

Comparison of the available genome assemblies for Asian seabass.

Category	This study	L. calcarifer (ASB-BC8)17
Genome survey (Mb)	576.74	593–648
Genome length (bp)	614,195,649	668,464,831
Longest scaffold (bp)	31,852,513	30,776,907
Number of scaffolds	24	3,807
Contig N50 (bp)	26,575,253	1,066,117
Scaffold N50 (bp)	26,575,253	25,848,596
GC content	40.7%	40.8%
BUSCO	100% (S:99.94%; D:0.16%)	99.7% (S:96%; D:3.7%)
Number of chromosomes	24	24
Chromosome length (bp)	614,195,649	586,924,032
Repetitive sequence	18.18%	/

Abbreviations: S, single copy complete genes; D, duplicated complete genes.

Identification of the centromere and telomere sequences

Telomeres were identified by searching for the target sequence (CCCTAA/TTAGGG) at both ends of each chromosome using Telomere-to-Telomere Toolkit quarTeT v1.1.1³³. Centromeres, as specialized DNA sequences connecting sister chromatids, exhibit complex structures in most animals and plants with highly repetitive satellite DNA and scattered retrotransposon sequences. In this study, after identifying repeat sequences according to TRF v4.0.4³⁴ and RepeatMasker v4.0.6³⁵ and obtaining a TE annotation file, quarTeT v1.1.1³³ was applied to identify centromeres, and the candidate interval range of every centromere was predicted. Ultimately, we determined that the Asian seabass genome contains a complete set of 24 centromeres and 48 telomeres (Table 3; Fig. 3).

Table 3.

Telomere and centromere positions in the assembled genome.

Chr	Contig	Length (bp)	Gap	Telomere (Te)				Centromere (Ce)
				Upstream Start	Upstream End	Downstream Start	Downstream End	Start	End
Chr01	1	31,852,513	0	251	3,343	31,848,788	31,852,328	1,505,150	1,784,594
Chr02	1	31,638,724	0	29	1,,056	31,638,090	31,638,401	4,132,572	4,235,565
Chr03	1	29,918,513	0	64	6134	29,917,458	29,918,485	27,923,600	28,634,341
Chr04	1	29,558,833	0	493	3,991	29,553,555	29,558,758	16,763,413	16,977,890
Chr05	1	29,570,087	0	38	7,538	29,569,431	29,570,087	28,554,835	29,493,014
Chr06	1	29,199,514	0	63	5,031	29,198,324	29,199,475	9,609	779,212
Chr07	1	29,179,243	0	5	3,891	29,179,155	29,179,208	28,437,506	28,968,124
Chr08	1	27,751,246	0	7	6,788	27,747,489	27,751,129	1,198,505	1,281,772
Chr09	1	27,635,561	0	3	5,138	27,632,115	27,635,202	24,779,176	24,919,240
Chr10	1	26,717,877	0	44	3,433	26,713,583	26,717,829	11,069,449	11,101,393
Chr11	1	26,575,253	0	102	4,914	26,568,413	26,575,161	23,814,253	24,022,695
Chr12	1	26,190,281	0	3	4,913	26,185,410	26,190,261	1,972,765	2,010,536
Chr13	1	25,913,521	0	2	2,370	25,908,749	25,913,302	23,680,651	23,817,342
Chr14	1	25,614,823	0	6	4,500	25,609,695	25,614,798	130,448	540,036
Chr15	1	25,420,547	0	26	3,571	25,420,129	25,420,291	80,051	483,348
Chr16	1	25,111,693	0	3	4,038	25,065,727	25,111,549	1,479,578	1,691,299
Chr17	1	23,846,329	0	304	4,318	23,841,449	23,846,329	20,554,022	20,584,804
Chr18	1	23,429,025	0	380	4,462	23,428,592	23,428,950	118,946	609,424
Chr19	1	22,557,243	0	558	3,151	22,550,858	22,557,068	1,711,822	1,903,333
Chr20	1	21,388,025	0	110	5,748	21,330,373	21,388,021	20,427,089	21,220,047
Chr21	1	21,383,011	0	29	5,745	21,378,941	21,382,968	20,010,214	20,126,177
Chr22	1	19,598,755	0	4	3,098	19,593,794	19,598,751	194,577	544,417
Chr23	1	19,288,435	0	148	3,373	19,285,242	19,288,431	17,836,318	17,972,547
Chr24	1	14,856,597	0	456	28,012	14,852,448	14,856,551	405,579	524,259

Fig. 3.

Genome-wide localization of repetitive elements (REs), telomeres and centromeres. The triangles at both ends of each chromosome represent the telomere regions, and the gully area within each chromosome stands for the centromere region.

Annotation of repeat elements

For prediction of repetitive elements (REs), tandem repeats were first annotated using TRF v4.0.4³⁴ and GMATA v2.2³⁶. TRF was employed to identify simple sequence repeats (SSRs), whereas GMATA was used to recognize all tandem REs across the entire genome.

Transposable elements (TEs) in the assembled genome were predicted using a combination of homology-based and de novo methods. For the homology approach, TEs were identified using RepeatMasker v4.0.6 and RepeatProteinMask v4.0.6³⁵. For the de novo approach, RepeatModeler v1.0.8³⁷ and LTR_FINDER v1.0.6³⁸ were employed to generate a de novo repeat library, and RepeatMasker was applied to annotate REs against this repeat library. The annotation results of all repetitive sequences were merged into a comprehensive dataset. This comprehensive annotation revealed 111.64 Mb of repetitive sequences, which account for 18.18% of the assembled Asian seabass genome (Fig. 3). The most abundant repetitive element was DNA transposons at 9.00% (55.26 Mb), followed by long interspersed nuclear elements (LINEs) at 2.89% (17.76 Mb) and long terminal repeats (LTRs) at 2.46% (15.07 Mb) (see Table 4).

Table 4.

Classification of repetitive sequences in Asian seabass genome.

Type			Length (bp)	Count	% of Genome
Dispersed repeats	DNA transposons		55,263,108	477,149	9.00
	Retroelements	LINE	17,763,761	124,264	2.89
		LTR	15,078,735	141,007	2.46
		SINE	2,414,481	20,229	0.39
	Unclassified		3,985,113	26,905	0.65
Tandem Repeats	Simple repeats		1,766,456	149,486	0.29
	Satellites		3,174,653	50,007	0.52
Unknown			12,194,948	95,844	1.98
Total			111,641,255	10,848,891	18.18

Prediction and functional annotation of protein-coding genes

Repetitive regions of the assembled genome were masked prior to prediction of genes and their structures. Protein-coding genes was annotated by combination of three methods, including de novo, homology and RNA-seq-based annotations. First, AUGUSTUS v3.2.1³⁹ and GlimmerHMM v3.0.4⁴⁰ were employed to perform the ab inito gene structure prediction. Second, GeMoMa v1.6.4⁴¹ was applied for the homology-based prediction. We aligned homology proteins from five representative fish species, including Epinephelus fuscoguttatus (brown-marbled grouper, GCA_011397635.1), Epinephelus moara (kelp grouper, GCA_006386435.1), Lates japonicus (Japanese lates, GCA_033238685.1), Perca flavescens (yellow Perch, GCA_004354835.1) and Sebastes umbrosus (Honeycomb rockfish, GCA_015220745.1) downloaded from the NCBI. Third, the RNA-seq data from seven tissues were assembled into contigs using Trinity v2.5.1⁴², and then gene structures were identified using PASA v2.3.3⁴³. Finally, gene sets were integrated by the Evidence Modeler (EVM) pipeline v1.0⁴⁴.

A total of 25,093 protein-coding genes were annotated, with an average gene length of 13.81 kb and an average coding sequence (CDS) length of 1,721.49 bp (Table 5). Protein-coding genes were evaluated using BUSCO with the actinopterygii_odb10 database as the reference. More than 98.8% of complete BUSCOs were identified within the predicted protein-coding genes.

Table 5.

Summary of the predicted gene structures using three methods.

Method	Software/Species	Number	Average length (bp)				Average exon per gene
			gene	CDS	exon	intron
De novo	Augustus	24,459	14,516.87	1,741.71	159.82	1,290.65	10.9
	Glimmer	39,727	14,042.91	1,020.96	161.29	2,443.21	6.33
Homolog	E. moara	50,987	23,273.19	1,658.37	180.14	2,634.1	9.21
	L. japonicus	51,010	19,380.37	1,656.83	179.59	2,154.73	9.23
	E. fuscoguttatus	52,748	23,530.9	1,656.82	180.53	2,674.84	9.18
	P. flavescens	53,847	24,587.81	1,638.19	181.46	2,858.73	9.03
	S. umbrosus	53,366	24,022.61	1,673.48	185.4	2,784.43	9.03
RNA-seq	PASA	21,217	16,814.96	3,699.28	302.27	1,167.04	12.24
Integrated	EVM	25,093	13,819.54	1,721.49	168.93	1,316.4	10.19

Functional annotation of the protein-coding genes was performed using Blastp v2.2.26⁴⁵, which aligned deduced protein sequences against five public databases including NCBI Non-Redundant Protein Sequence (NR), SwissProt⁴⁶, Gene Ontology (GO)⁴⁷, Kyoto Encyclopedia of Genes and Genomes (KEGG)⁴⁸ and EuKaryotic Orthologous Groups (KOG)⁴⁹, with an E-value cutoff of <1e−5. Ultimately, 23,711 protein-coding genes (94.49% of the total predicted genes) were functionally annotated, with at least one hit for each gene in the searched databases (Table 6).

Table 6.

Functional annotation of predicted protein-coding genes.

Database	Number	Percentage (%)
Total	25,093	100
NR	23,699	94.44
Swissprot	21,269	84.76
KEGG	16,838	67.10
GO	16,260	64.80
KOG	15,510	61.81
Overall	23,711	94.49

Overall represents the total number of annotated genes with at least one hit from the five searched databases.

Data Records

Files of the MGI, PacBio, ONT, Hi-C and transcriptome sequencing, and the assembled genome for Asian seabass were deposited at NCBI under the accession number PRJNA1245135. Raw reads are available in the Sequence Reads Archive (SRA) with the accession numbers SRR32997291 to SRR32997305⁵⁰. The genome assembly, predicted coding sequences and function annotation files of Asian seabass were stored in Figshare (No: m9.figshare.28735226)⁵¹. The genome assembly has also been deposited at the NCBl/GenBank under the accession number of GCA_051027255.1⁵².

Technical Validation

To evaluate the quality of our genome assembly, we employed four approaches. First, BUSCO v5.2.2⁵³ was employed to examine completeness. A total of 100% (single copy complete genes (S): 99.84%, duplicated complete genes (D): 0.16%) of complete BUSCOs in the actinopterygii_odb10 database were identified. Second, Merqury v1.328⁵⁴ was applied to estimate the base-level accuracy and completeness on the basis of k-mer counts (generated from Illumina and PacBio HiFi reads), resulting in a QV of 40.59 and 57.80 respectively. Third, Clipping information for Revealing Assembly Quality (CRAQ, v1.09)⁵⁵ was used to assess the accuracy of our genome assembly based on PacBio HiFi and Illumina reads, resulting in a R-AQI (assembly quality indicator) of 98.42 and a S-AQI of 99.45. Fourth, we mapped the sequencing data to the assembled genome using bwa v0.7.17⁵⁶ and minimap2 v2.26⁵⁷, which showed mapping rates of 99.46% for the MGI data, 99.99% for the PacBio data, and 98.43% for the ONT data. These results collectively support high quality of the Asian seabass genome assembly. The BUSCO completeness value was calculated to be 98.8% for the predicted protein-coding genes of Asian seabass (Table 7). To further evaluate the quality of these predicted protein-coding genes, we aligned the transcriptome data to the assembled genome using STAR v 2.7.11b⁵⁸, and then calculated the exonic coverage rate with bedtools v2.29.2⁵⁹. We observed that 94.71% of the exonic regions had been covered with sequencing reads, indicating high annotation accuracy (see Table 7).

Table 7.

Assessment metrics of the genome assembly and annotation.

Type	Evaluation Methods		Results
Genome accuracy and completeness	Mapping short reads rate		99.46%
	Mapping HiFi reads rate		99.99%
	Mapping ONT reads rate		98.43%
	QV	Short reads	40.59
		HiFi reads	57.80
	CRAQ	R-AQI	98.42%
		S-AQI	99.45%
	BUSCO		100%
Annotation quality	Complete BUSCOs		98.8% (3,599)
	Complete and single-copy BUSCOs (S)		98.2% (3,576)
	Complete and duplicated BUSCOs (D)		0.6% (23)
	Fragmented BUSCOs (F)		0 (0)
	Missing BUSCOs (M)		1.2% (41)
	RNA-seq coverage ratio of the exonic regions		94.71%

Author contributions

Q.S. conceived and designed the study. X.Z., J.W. and J.C. collected the samples. X.Z., J.C. and J.W. performed data analysis. J.W. and W.Z. conducted experiments for species identification. X.Z. and J.W. wrote the manuscript. Q.S. revised the manuscript. All authors read and approved the final manuscript for publication.

Code availability

The versions and parameters of bioinformatics tools applied in this study have been described in the Method section. If no parameter is provided, the default is set. No custom code was used.

Competing interests

The authors declare no competing interests.