Skip to main content
NCBI home page
GigaByte logoLink to GigaByte
. 2021 Apr 1;2021:gigabyte17. doi: 10.46471/gigabyte.17

Chromosome-level genome assembly of the humpback puffer, Tetraodon palembangensis

Rui Zhang 1,†, Chang Li 1,†, Mengjun Yu 1,†, Xiaoyun Huang 1, Mengqi Zhang 1, Shanshan Liu 1, Shanshan Pan 1, Weizhen Xue 1, Congyan Wang 1, Chunyan Mao 1, He Zhang 1,2,*, Guangyi Fan 1,*
PMCID: PMC9632004  PMID: 36824331

Abstract

The humpback puffer, Tetraodon palembangensis, is a poisonous freshwater pufferfish species mainly distributed in Southeast Asia (Thailand, Laos, Malaysia and Indonesia). The humpback puffer has many interesting biological features, such as inactivity, tetrodotoxin production and body expansion. Here, we report the first chromosome-level genome assembly of the humpback puffer. The genome size is 362 Mb, with a contig N50 value of ∼1.78 Mb and a scaffold N50 value of ∼15.8 Mb. Based on this genome assembly, ∼61.5 Mb (18.11%) repeat sequences were identified, 19,925 genes were annotated, and the function of 90.01% of these genes could be predicted. Finally, a phylogenetic tree of ten teleost fish species was constructed. This analysis suggests that the humpback puffer and T. nigroviridis share a common ancestor 18.1 million years ago (MYA), and diverged from T. rubripes 45.8 MYA. The humpback puffer genome will be a valuable genomic resource to illustrate possible mechanisms of tetrodotoxin synthesis and tolerance.

Data description

Background and context

The humpback puffer, Tetraodon palembangensis (NCBI Taxonomy ID: 1820603, Fishbase ID: 25179), also known as Pao palembangensis, is widely distributed in Southeast Asia and prefers to live in alkalescent, warm (24–28°), and slow-flowing rivers (Figure 1a) [1]. The female and male humpback puffers have a similar body size, but the male’s rear hump is much bigger than that of the female [2]. The humpback puffer is a popular ornamental fish because of its beautiful skin colouration and patterns. Unlike other species of predatory pufferfish, the humpback puffer is lazy and will not initiatively look for food [1]. Furthermore, its body contains a deadly toxin, known as tetrodotoxin (TTX), and it can swell up to three times its normal size as a defense mechanism when threatened [1]. Previous studies have shown that the toxicity of the humpback puffer varies greatly between seasons [3]. The wild population of humpback puffer has declined in recent years owing to the destruction of suitable habitat caused by climate change and overfishing [4].

Figure 1.

Figure 1.

Open in a new tab

Tetraodon palembangensis. (a) Photograph of Tetraodon palembangensis. Photo courtesy of Preston Swipe, Aquatic Arts LLC. (b) The 17-mer depth distribution of the stLFR reads. The estimated genome size is 385 Mb; heterozygosis is 0.21%.

With these biological characteristics and a small genome size, the humpback puffer is an ideal species for genetic study [5]. It is also a species in the Fish10K Genome Project, a subproject of the Earth BioGenome Project, which aims to sample, sequence, assemble and analyse the genomes of 10,000 fish species [6]. In this study, we provide a chromosome-scale genome assembly of the humpback puffer. This assembly will be valuable for further study of mechanisms, such as tetrodotoxin synthesis and expansion defense. Comparative genomic analysis will help us to better understand the phylogenetic evolution and special gene families of the Tetraodontidae.

Methods

All methods used to isolate DNA/RNA, construct libraries, and conduct genomic sequencing are available in a protocols.io collection (Figure 2 [7]).

Figure 2.

Figure 2.

Open in a new tab

Protocols.io collection for the chromosome-level genome assembly of the humpback puffer, Tetraodon palembangensis. https://www.protocols.io/widgets/doi?uri=dx.doi.org/10.17504/protocols.io.bs8inhue

Sample collection and sequencing

The sample (CNGB ID: CNS0224034) used in this study was an adult humpback puffer bought from the YueHe Flower-Bird-Fish market in Guangzhou Province, China. DNA and RNA were both isolated from blood following published protocols [8, 9]. Then, a paired-end single tube long fragment reads (stLFR) library and an RNA library were constructed according to the protocol published by Wang et al. [10]. A Hi-C library was constructed from blood according to the protocol published by Huang et al. [11]. These three libraries were then sequenced on the BGISEQ-500 platform (RRID:SCR_017979) [12]. A Nanopore library was constructed with DNA isolated from blood using the QIAamp DNA Mini Kit (Qiagen) [13] and sequenced on the GridION platform (RRID:SCR_017986) [14]. In total, we obtained 146 Gb (∼312×) raw stLFR data, 21 Gb raw RNA data, 19 GB (∼49×) raw Hi-C data, and 12 GB (∼32×) raw Nanopore data (Table 1).

Table 1.

Statistics of sequencing data.

Libraries Read lengths (bp) Raw data Valid data
Total bases (Gb) Sequencing depth (×) Total bases (Gb) Sequencing depth (×)
stFLR PE100 120.4 311.69 62.6 162.60
RNA PE 100 20.6 – 20.0 –
Hi-C PE 100 19.01 49.43 5.4 14.04
Nanopore CN50: 32 kb 12.3 31.98 – –
Open in a new tab

Sequencing depth = total bases/genome size, where the genome size is the result of k-mer estimation in Table 2. Abbreviations: CN5, contig N50; PE, paired end.

Raw stLFR reads were subjected to quality control to improve the assembly quality. Firstly, we obtained co-barcoding information from the last 42 bases of read 1 and deleted the last 42 bases. Then SOAPnuke (v1.6.5, RRID:SCR_015025) [15] was used to filter remaining reads, using the parameters: “–M 1 –d –A 0.4 –n 0.05 –l 10 –q 0.4 –Q 2 –G –5 0” [16]. Finally, 62 Gb (152×) clean data were retained for further assembly. Raw RNA reads were also filtered by SOAPnuke using the parameters: “–M 1 –A 0.4 –n 0.05 –l 10 –q 0.4 –Q 2 –G –5 0”, generating 20 Gb clean data. Raw Hi-C data were produced with a quality control using HiC-Pro (v. 2.8.0) [16] with default parameters. This generated 5.4 Gb validated data, which accounted for 28.81% of all data (Table 1).

Genome assembly

Jellyfish (v2.2.6, RRID:SCR_005491) was used to count k-17mers of all clean stLFR reads [17]. Genomescope [18] was used to estimate the humpback puffer genome size at about 385 Mb (Table 2 and Figure 1b).

Table 2.

Statistics of 17-mer analysis.

k-mer k-mer number k-mer depth (×) Heterozygosity (%) Genome size (bp)
17 48,458,703,762 126 0.205 384,592,887
Open in a new tab

The genome size, G, was defined as G = Knum∕Kdepth, where the Knum is the total number of k-mers, and Kdepth is the most frequently occurring frequency.

To assemble the humpback puffer genome, we firstly converted the format of clean stLFR reads, then used Supernova (v. 2.0.1, RRID:SCR_016756) to perform the draft assembly. Then, we used GapCloser (v. 1.12, RRID:SCR_015026) [19] to fill gaps with stLFR reads. To futher improve the assembly quality, TGSgapFiller [20] was then used to re-fill gaps with Nanopore reads, and Pilon (v. 1.22, RRID:SCR_014731) [21] was used to polish the assembly twice. At this stage, the genome assembly was about 362 Mb, with 7.1-Mb scaffold N50 and 1.8-Mb contig N50 values (Table 3).

Table 3.

Statistics of the draft assembly of the humpback puffer genome.

Statistics Scaffold Contig
Total number (#) 5291 6190
Total length (bp) 361,704,206 360,427,744
Gap (N) (bp) 1,276,462 0
Average length (bp) 68,362 58,227
N50 length (bp) 7,059,990 1,830,664
N90 length (bp) 453,057 157,209
Maximum length (bp) 19,534,197 9,842,180
Minimum length (bp) 682 48
GC content (%) 44.66 44.66
Open in a new tab

With the genome and validated Hi-C data from HiC-Pro, the contact matrix was generated by Juicer (v3, RRID:SCR_017226). Finally, we perfomed chromosomal-level scaffolding using the 3D de novo assembly (3D-DNA) pipeline (v. 170123) [22]. This anchored 91.2% of all sequences to 18 chromosomes, with a length ranging from 11 Mb to 35 Mb (Table 4 and Figure 3).

Table 4.

Statistics of the Hi-C scaffolding of the humpback puffer genome.

Statistics Scaffold Contig
Total number (#) 5366 6435
Total length (bp) 361,698,760 360,427,744
Gap (N) (bp) 1,271,016 0
Average length (bp) 67,406 56,011
N50 length (bp) 15,808,960 1,794,775
N90 length (bp) 11,014,520 117,115
Maximum length (bp) 34,916,285 9,792,502
Minimum length (bp) 682 48
GC content (%) 44.66 44.66
Open in a new tab

Figure 3.

Figure 3.

Open in a new tab

Heat map of chromosomal interaction from Hi-C reads. Grey lines show the border between chromosomes.

The karyotype differs among genuses in Tetraodontidae [23–27]. For example, Takifugu rubripes, Takifugu obscurus and Takifugu flavidus have 2n = 44 chromosomes, while Tetraodon nigroviridis and Tetraodon fluviatilis have 2n = 42 chromosomes. In addition, Thamnaconus septentrionalis (Monacanthidae) has 2n = 40 chromosomes. Thus, we defined the chromosome number of the humpback puffer to be 18, according to the apparent and logical interactions by Hi-C reads.

Genomic annotation

Two methods were used to annotate repetitive sequences. Firstly, we aligned the genome to the Repbase library by TRF (v.4.09) [28]. RepeatMasker (v. 3.3.0, RRID:SCR_012954) and RepeatProteinMask (v. 3.3.0) [29] were then used to predict and classify the repetitive sequences. Secondly, we constructed a repeat library using RepeatModeler (v1.0.8, RRID:SCR_015027) and classified the transposable elements (TEs) with RepeatMasker (v. 3.3.0) [29]. The results of both methods were amalgamated to give a total of 65 Mb repeat sequences and 59 Mb TEs, accounting for 18.11% (Table 5 and Figure 4a) and 16.62% of the entire genome, respectively (Table 6 and Figure 4a).

Table 5.

Statistics of repeat sequences.

Type Repeat size (bp) % of genome
TRF 9,050,571 2.52
RepeatMasker 34,142,529 9.50
RepeatProteinMask 17,674,660 4.92
De novo 57,492,865 16.00
Total 65,080,476 18.11
Open in a new tab

Figure 4.

Figure 4.

Open in a new tab

Annotation of the Tetraodon palembangensis genome. (a) Basic genomic elements of the genome. LTR, long terminal repeat; LINE, long interspersed nuclear elements; SINE, short interspersed elements. (b) Physical map of mitochondrial assembly.

Table 6.

Statistics of transposable elements (TEs).

Type RepBase TEs TE proteins De novo Combined TEs
Length (bp) % of genome Length (bp) % of genome Length (bp) % of genome Length (bp) % of genome
DNA 12,412,491 3.45 1,086,262 0.30 16,089,219 4.48 22,470,373 6.25
LINE 18,430,929 5.13 13,695,154 3.81 29,418,621 8.19 33,421,782 9.30
SINE 524,061 0.15 0 0.00 289,252 0.08 789,086 0.22
LTR 5,393,600 1.50 2,906,451 0.81 12,758,934 3.55 15,803,098 4.40
Other 8,290 0.00 228 0.00 0 0.00 8,518 0.00
Unknown 0 0.00 0 0.00 3,202,764 0.89 3,202,764 0.89
Total 34,142,529 9.50 17,674,660 4.92 55,052,617 15.32 59,729,335 16.62
Open in a new tab

Abbreviations: LINE, long interspersed nuclear elements; LTR, long terminal repeats; SINE, short interspersed nuclear elements; TE, transposable elements.

Using the clean, reformatted stLFR reads, the mitochondrial genome of the humpback puffer was assembled using MitoZ [30] with default parameters. Mitochondrial genes were annotated using the MitoAnnotator tool of the mitofish pipeline [31] (Figure 4b). For gene structural annotation, we performed de novo prediction using AUGUSTUS (v3.1, RRID:SCR_008417) [32], GlimmerHMM (v3.0.4, RRID:SCR_002654) [33], and Genscan (RRID:SCR_013362) [34]. We also used TRINITY (v2.8.5, RRID:SCR_013048) [35] to assemble a draft transcriptome with clean RNA reads, then HISAT2 (v2.1.0, RRID:SCR_015530)-StringTie (v1.3.4, RRID:SCR_016323) [36] and PASA (v2.3.3, RRID:SCR_014656)-TransDecoder (RRID:SCR_017647) [37] to predict transcripts. GeneWise (v2.4.1, RRID:SCR_015054) [38] was used for homologous annotation, with protein data obtained from the National Center for Biotechnology Information (NCBI) database for the following eight species: Danio rerio (NCBI, GenBank ID:50), Cynoglossus semilaevis (NCBI, GenBank ID:11788), Gasterosteus aculeatus (NCBI, GenBank ID:146), Gadus morhua (NCBI, GenBank ID:2661), Larimichthys crocea (NCBI, GenBank ID:12197), Oreochromis niloticus (NCBI, GenBank ID:197), Oryzias latipes (NCBI, GenBank ID:542), and Takifugu rubripes (NCBI, GenBank ID:63). Finally, these three types of evidence were integrated using EVidenceModeler (v1.1.1, RRID:SCR_014659) [39], generating 19,925 nonredundant coding genes, each containing an average of 11 exons and a 1945 bp coding region (Table 7).

Table 7.

Statistics of the predicted genes in the humpback puffer genome.

Gene set Gene number Average transcript length (bp) Average CDS length (bp) Average intron length (bp) Average exon length (bp) Average exons per gene
Homolog Cynoglossus semilaevis 19,686 9136.12 1715.14 856.1 177.4 9.67
Danio rerio 19,348 15,066.80 1577.39 1718.92 178.28 8.85
Gadus morhua 20,361 7040.85 1441.77 744.62 169.23 8.52
Gasterosteus aculeatus 26,630 6896.85 1474.53 686.88 165.79 8.89
Larimichthys crocea 21,220 9425.27 1690.06 902.2 176.53 9.57
Oreochromis niloticus 24,562 9494.62 1789.15 829.18 173.82 10.29
Oryzias latipes 23,332 8859.46 1467.62 962.5 169.08 8.68
Takifugu rubripes 19,635 7762.47 1645.04 707.22 170.47 9.65
De novo Augustus 21,662 7149.08 1725.00 659.42 186.98 9.23
Genscan 25,933 9855.53 1791.43 990.72 196.01 9.14
GlimmerHMM 99,722 1192.96 594.53 378.42 230.31 2.58
Transcript Pasa & Transdecoder 33,965 4856.71 1186.88 558.33 156.73 7.57
Hisat & Stringtie 31,664 5551.59 1303.52 608.39 163.3 7.98
EVM 19,925 9418.80 1945.48 757.65 179.08 10.86
Open in a new tab

The EVM gene set contains the integrated result of De novo gene predictions, homolog gene predictions and transcript annotation by EVM software.

For gene function annotation, we aligned the 19,925 genes to the TrEMBL (UniProtKB, RRID:SCR_004426) [40], Swissprot [41], Kyoto Enyclopedia of Genes and Genomes (KEGG, RRID:SCR_012773) [42], Gene Ontology (GO, RRID:SCR_002811) [43] and InterProScan (RRID:SCR_005829) [44] databases. Overall, 90.1% of all genes were able to be functionally annotated (Table 8 and Figure 3).

Table 8.

Statistics of the functional annotation.

Database Number of genes Gene functionally annotated (%)
Total 20,057 100.00
SwissProt 17,333 86.42
KEGG 16,182 80.68
TrEMBL 18,037 89.93
Interpro 17,108 85.30
Overall 18,064 90.06
Open in a new tab

Genome evolution

To study the evolutionary status of humpback puffer among bony fish species, we clustered gene families by alignment using protein sequences of the humpback puffer and nine other teleosts (Xiphophorus maculatus, Gasterosteus aculeatus, Sebastes schlegelii, Oryzias latipes, Gadus morhua, Oreochromis niloticus, Tetraodon nigroviridis, Danio rerio, and Takifugu rubripes) using the TreeFam v0.50 pipeline [45]. Protein-coding genes sequences for all of these species were downloaded from NCBI, except for S. schlegelii  [46], which was obtained from the China National Genebank Nucleotide Sequence Archive (CNSA; Accession ID: CNP0000222). To improve analysis quality, we removed genes either with frameshifts, or less than 50 amino acids, as well as redundant copies, only keeping the longest transcripts for comparative genomic analysis. A total of 21,022 gene families were identified, of which 40 gene families were unique to the humpback puffer (Table 9 and Figure 5a).

Table 9.

Statistics of gene family clustering.

Species Total number of genes Number of unclustered genes Number of gene families Number of unique families Average number of genes per family
D. rerio 30,067 2171 18,635 735 1.5
G. aculeatus 20,756 728 15,995 11 1.25
G. morhua 19,987 525 15,650 11 1.24
S. schlegelii 24,094 558 16,991 30 1.39
O. latipes 19,535 984 14,873 71 1.25
O. niloticus 21,431 160 15,811 13 1.35
T. nigroviridis 19,544 805 14,916 50 1.26
T. palembangensis 19,796 690 15,830 40 1.21
T. rubripes 18,459 207 14,733 6 1.24
X. maculatus 20,356 271 16,446 3 1.22
Open in a new tab

Figure 5.

Figure 5.

Open in a new tab

Comparative analysis of the Tetraodon palembangensis and nine teleosts. (a) Clustering of gene families. (b) Phylogenetic tree constructed with the single-copy gene families. The fossil correction nodes in the tree are highlighted by red dots.

Of all 21,022 gene families, we identified 4461 single-copy protein-coding genes shared by all species. We used MUSCLE v3.8.31 [47] to align these orthologs, with default parameters. Then, the alignments were concatenated into a 3,584,782 amino acid “super alignment matrix”. Based on this matrix, a phylogenetic tree was constructed using RAxML v8.2.4 [48], with the best amino acid substitution model-JTT. Clade support was assessed using a bootstrapping algorithm with 1000 alignment replicates (Figure 5b). Next, we calculated the divergence time among these teleosts using the MCMCTree tool included in PAML (v4.7a, RRID:SCR_014932) [49], with parameters of “–rootage 500 -clock 3 -alpha 0.431879”. The fossil correction time (Table 10) was obtained from Timetree [50]. The result showed that the humpback puffer and T. nigroviridis, two species belonging to the same genus, shared a common ancestor 18.1 millon years ago (MYA) and diverged from T. rubripes 18.1 MYA (Figure 5b).

Table 10.

Fossil correction time used in divergence analysis.

Taxon 1 Taxon 2 Fossil time (MYA) Minimum (MYA) Maximum (MYA)
Takifugu rubripes Tetraodon nigroviridis 52 42 59
Takifugu rubripes Gadus morhua 148 141 170
Oryzias latipes Xiphophorus maculatus 93 76 111
Oryzias latipes Gasterosteus aculeatus 128 105 154
Oryzias latipes Danio rerio 229.9 204.5 255.3
Open in a new tab

Data validation and quality control

To demonstrate the quality of genome assembly and gene set, we performed a qulity evaluation using the actinopterygii_odb10 database from Benchmarking Universal Single-Copy Orthologs (BUSCO v.4.1.2, RRID:SCR_015008) [51]. The results showed that 95.7% and 90.7% complete BUSCOs were covered by the genome assembly and gene set, respectively (Table 11).

Table 11.

Statistics of the BUSCO assessment.

Types of BUSCOs Genome assembly Gene set
Number Percentage (%) Number Percentage (%)
Complete BUSCOs 3486 95.7 3303 90.7
Complete and single-copy BUSCOs 3427 94.1 3252 89.3
Complete and duplicated BUSCOs 59 1.6 51 1.4
Fragmented BUSCOs 45 1.2 96 2.6
Missing BUSCOs 109 3.1 241 6.7
Total BUSCOs groups searched 3640 100 3640 100
Open in a new tab

Reuse potential

We assembled the first annotated chromosome-level genome of the humpback puffer. These resources will be helpful to study the mechanism of body expansion displayed by this fish species, the synthesis mechanism and treatment of tetrodotoxin, as well as the evolution of freshwater puffer. Futhermore, the humpback puffer genome will fill a gap missing from the Fish 10K program and in the phylogenetic tree of life.

Acknowledgements

We thank for the China National Genebank for technical support in constructing and sequencing the stLFR library.

Funding Statement

This work was supported by the special funding of “Blue granary” scientific and technological innovation of China (2018YFD0900301-05).

Data availability

We have deposited the project at CNGB Nucleotide Sequence Archive (CNSA) where the accession ID is CNP0001025. The genomic data can be obtained in GigaScience Database [52]. The sequencing data have been deposited at National Center for Biotechnology Information (NCBI) where the bioproject accession ID is PRJNA597275.

Declarations

List of abbreviations

bp: base pair; BUSCO: Benchmarking Universal Single-Copy Orthologs; CNSA: China National Gene Bank Nucleotide Sequence Archive; Gb: gigabase; GO: Gene Ontology; kb: kilobase; KEGG: Kyoto Enyclopedia of Genes and Genomes; Mb: megabase; ML: Maximum Likelihood; NCBI: National Center for Biotechnology Information; stLFR: single tube long fragment reads; TE: transposable element.

Ethical approval

All resources used in this study were approved by the Institutional Review Board of BGI (IRB approval No. FT17007). This experiment has passed the ethics audit of Beijing Genomics Institute (BGI) Gene Bioethics and Biosecurity Review Committee.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the special funding of “Blue granary” scientific and technological innovation of China (2018YFD0900301-05).

Authors’ Contributions

H.Z. and G.F. designed this project. M.Z. prepared the samples. S.L., S.P., W.X., C.W. and C.M. conducted the experiments. R.Z., C.L., M.Y. and X.H. did the analyses. R.Z., C.L. and M.Y. wrote and revised the manuscript. All authors read and approved the final version of the manuscript.

References

  • 1.Saitanu K, et al. Toxicity of the freshwater puffer fish Tetraodon fangi and T. palembangensis from Thailand. Toxicon, 1991; 29: 895–897. [DOI] [PubMed] [Google Scholar]
  • 2.Subamia IW, Sudarto S, Purbowasito W, . Sex determination in Indonesian pufferfish Tetraodon palembangensis Bleeker, 1852: implication for aquaculture and conservation. Indones. Aquac. J., 2011; 6: 37–45. [Google Scholar]
  • 3.Jaillon O, et al. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature, 2004; 431: 946–957. [DOI] [PubMed] [Google Scholar]
  • 4.Sutaria D, Panicker D, Jog K, Sule M, Muralidharan R, Bopardikar I, . Humpback dolphins (Genus Sousa) in India: an overview of status and conservation issues. Adv. Marine Biol., 2015; 72: 229–256. [DOI] [PubMed] [Google Scholar]
  • 5.Hedges SB, . The origin and evolution of model organisms. Nat. Rev. Genet., 2002; 3: 838–849. [DOI] [PubMed] [Google Scholar]
  • 6.Fan G, et al. Initial data release and announcement of the 10,000 Fish Genomes Project (Fish10K). GigaScience, 2020; 9: giaa080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Zhang R, et al. Protocols for “Chromosome-level genome assembly of the humpback puffer, Tetraodon palembangensis”. protocols.io. 2021; 10.17504/protocols.io.bs8inhue. [DOI] [PMC free article] [PubMed]
  • 8.Chang L, . Hi-C library preparation for the Lateolabrax maculatus genome. protocols.io. 2018. l; 10.17504/protocols.io.ss4eegw. [DOI]
  • 9.Julkowska M, . Protocols for “RNA isolation with TRIzol”. protocols.io. 2018; 10.17504/protocols.io.pbndime. [DOI]
  • 10.Wang O, et al. Efficient and unique cobarcoding of second-generation sequencing reads from long DNA molecules enabling cost-effective and accurate sequencing, haplotyping, and de novo assembly. Genome Res., 2019; 29: 798–808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Huang J, et al. BGISEQ-500 WGS library construction. protocols.io. 2018; 10.17504/protocols.io.ps5dng6. [DOI]
  • 12.Huang J, . BGISEQ-500 sequencing. protocols.io. 2018; 10.17504/protocols.io.pq7dmzn. [DOI]
  • 13.Pederson ER, . RNA and DNA extraction (Qiagen) of frozen tissue to nanopore sequencing. protocols.io. 2021; 10.17504/protocols.io.bj7tkrnn. [DOI]
  • 14.Zhang R, . Oxford Nanopore sequencing and library construction. protocols.io. 2021; 10.17504/protocols.io.btignkbw. [DOI]
  • 15.Chen Y, et al. SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data. Gigascience, 2018; 7: gix120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Servant N, et al. HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. Genome Biol., 2015; 16: 259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Li R, et al. The sequence and de novo assembly of the giant panda genome. Nature, 2010; 463: 311–317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Vurture GW, et al. GenomeScope: fast reference-free genome profiling from short reads. Bioinformatics, 2017; 33: 2202–2204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Luo R, et al. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience, 2012; 1: 18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Xu M, Guo L, Gu S, Wang O, Zhang R, Peters BA, Fan G, Liu X, Xu X, Deng L, Zhang Y, . TGS-GapCloser: A fast and accurate gap closer for large genomes with low coverage of error-prone long reads. Gigascience, 2020; 9(9): giaa094. doi: 10.1093/gigascience/giaa094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Walker BJ, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PloS One, 2014; 9: e112963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Liu X, . Protocols for “the pipeline of Hi-C assembly”. protocols.io. 2018; 10.17504/protocols.io.qradv2e. [DOI]
  • 23.Wataru K, et al. Integration of the genetic map and genome assembly of fugu facilitates insights into distinct features of genome evolution in teleosts and mammals. Genome Biol. Evol., 2011; 3: 424–442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kang S, et al. Chromosomal-level assembly of Takifugu obscurus (Abe, 1949) genome using third-generation DNA sequencing and Hi-C analysis. Mol. Ecol. Resour., 2020; 20: 520–530. [DOI] [PubMed] [Google Scholar]
  • 25.Zhou Y, et al. Chromosome genome assembly and annotation of the yellowbelly pufferfish with PacBio and Hi-C sequencing data. Sci. Data, 2019; 6: 267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Mandrioli M, Cuoghi B, Marini M, Manicardi GC, . Cytogenetic analysis of the pufferfish Tetraodon fluviatilis (Osteichthyes). Chromosome Res., 2000; 8: 237. [DOI] [PubMed] [Google Scholar]
  • 27.Li F, et al. Morphological structure and karyotype of Thamnaconus septentrionalis. South China Fisheries Science, 2019; 15: 104–112. [Google Scholar]
  • 28.Benson G, . Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res., 1999; 27: 573–580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Tarailo-Graovac M, Chen N, . Using RepeatMasker to identify repetitive elements in genomic sequences. Curr. Protoc. Bioinformatics, 2009; 25: 4.10.1–4.10.14. [DOI] [PubMed] [Google Scholar]
  • 30.Meng G, Li Y, Yang C, Liu S, . MitoZ: a toolkit for animal mitochondrial genome assembly, annotation and visualization. Nucleic Acids Res., 2019; 47: e63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Wataru I, et al. MitoFish and MitoAnnotator: a mitochondrial genome database of fish with an accurate and automatic annotation pipeline. Mol. Biol. Evol., 2013; 30: 2531–2540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Stanke M, Keller O, Gunduz I, Hayes A, Waack S, Morgenstern B, . AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Res., 2006; 34(suppl_2): W435–W439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Majoros WH, Pertea M, Salzberg SL, . TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders. Bioinformatics, 2004; 20: 2878–2879. [DOI] [PubMed] [Google Scholar]
  • 34.Burge C, Karlin S, . Prediction of complete gene structures in human genomic DNA. J. Mol. Biol., 1997; 268: 78–94. [DOI] [PubMed] [Google Scholar]
  • 35.Grabherr MG, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol., 2011; 29: 644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL, . Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat. Protoc., 2016; 11: 1650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Campbell MA, Haas BJ, Hamilton JP, Mount SM, Buell CR, . Comprehensive analysis of alternative splicing in rice and comparative analyses with Arabidopsis. BMC Genomics, 2006; 7: 327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Doerks T, Copley RR, Schultz J, Ponting CP, Bork P, . Systematic identification of novel protein domain families associated with nuclear functions. Genome Res., 2002; 12: 47–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Haas BJ, et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biol., 2008; 9: R7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Elsik CG, Mackey AJ, Reese JT, Milshina NV, Roos DS, Weinstock GM, . Creating a honey bee consensus gene set. Genome Biol., 2007; 8: R13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Bairoch A, Apweiler R, . The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Res., 2000; 28: 45–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Kanehisa M, Goto S, . KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res., 2000; 28: 27–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Harris MA, Clark J, Ireland A, Lomax J, Ashburner M, Foulger R, . The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res., 2004; 32(Suppl_1): D258–D261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Jones P, et al. InterProScan 5: genome-scale protein function classification. Bioinformatics, 2014; 30: 1236–1240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Li H, et al. TreeFam: a curated database of phylogenetic trees of animal gene families. Nucleic Acids Res., 2006; 34: D572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.He Y, et al. A chromosome level genome of black rockfish, Sebastes schlegelii, provides insights into the evolution of live birth. Mol. Ecol. Resour., 2019; 19: 1309–1321. [DOI] [PubMed] [Google Scholar]
  • 47.Edgar RC, . MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res., 2004; 19(32): 1792–1797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Stamatakis A, . RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 2006; 22: 2688–2690. [DOI] [PubMed] [Google Scholar]
  • 49.Yang Z, . PAML: a program package for phylogenetic analysis by maximum likelihood. Bioinformatics, 1997; 13: 555–556. [DOI] [PubMed] [Google Scholar]
  • 50.Sudhir K, Stecher G, Suleski M, Hedges SB, . TimeTree: a resource for timelines, timetrees, and divergence times. Mol. Biol. Evol., 2017; 34: 1812. [DOI] [PubMed] [Google Scholar]
  • 51.Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM, . BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics, 2015; 31: 3210–3212. [DOI] [PubMed] [Google Scholar]
  • 52.Zhang R, et al. Genome data for the chromosome-level assembly of the humpback puffer, Tetraodon palembangensis. GigaScience Database. 2020; 10.5524/100755. [DOI] [PMC free article] [PubMed]
GigaByte. 2021 Apr 1;2021:gigabyte17.

Article Submission

Rui Zhang
GigaByte.

Assign Handling Editor

Editor: Scott Edmunds
GigaByte.

Review MS

Editor: Sven Winter
Upload additional files DRR-202011-01/form/gx-DR-1604540547_SW.pdf
Reviewer name and names of any other individual's who aided in reviewer Sven Winter
Do you understand and agree to our policy of having open and named reviews, and having your review included with the published papers. (If no, please inform the editor that you cannot review this manuscript.) Yes
Is the language of sufficient quality? No
Please add additional comments on language quality to clarify if needed In general, most of the manuscript is written in a sufficient quality, but there are certain parts that need improvement. Please see detailed comments below.
Are all data available and do they match the descriptions in the paper? No
Additional Comments The data under the listed BioProject PRJNA597275 is not the same as described in the manuscript. I would suggest, that the authors update the species name on NCBI and make sure that the pacbio data is uploaded (so far I only see Nanopore data). The amount of stLFR data is also more than described in the manuscript.
Are the data and metadata consistent with relevant minimum information or reporting standards? See GigaDB checklists for examples <a href="http://gigadb.org/site/guide" target="_blank">http://gigadb.org/site/guide</a> Yes
Additional Comments
Is the data acquisition clear, complete and methodologically sound? No
Additional Comments It mostly is but it needs to be verified that pacbio and not ONT data was used.
Is there sufficient detail in the methods and data-processing steps to allow reproduction? No
Additional Comments I would like to see more details about the library preparations. Even though the authors reference protocols, basic information, e.g., what tissue type was used for Hi-C, should be given in the manuscript. Also again, the issue with pacbio or ONT needs to be resolved, and details about either library preparation need to be specified (pacbio CLR or CCS?, ONT libprep kit and sequencer)
Is there sufficient data validation and statistical analyses of data quality? Yes
Additional Comments I assume that there is, but I would like to see a bit more details of how the data was filtered, and the quality was checked. For example, how exactly did SOAPnuke filter the stLFR reads? What is an obvious sequencing error rate? Why was 50% of the data lost in the process? Same for the Hi-C data, why was there so much loss of raw data?
Is the validation suitable for this type of data? Yes
Additional Comments Yes I think it is, but it needs to be explained more. See detailed comments below.
Is there sufficient information for others to reuse this dataset or integrate it with other data? Yes
Additional Comments
Any Additional Overall Comments to the Author Overall, this is a good genome assembly and the manuscript is suitable for publication in Gigabyte, yet there are some methods that need to be described in more detail to be reproducible. I hope that my comments will help the authors to improve the readability and completeness of the manuscript. As page and line numbers are missing, it was easier to add my comments directly to the manuscript file. Therefore, please find my detailed comments attached. Looking forward to seeing this manuscript published in Gigabyte soon. Sven Winter
Recommendation Major Revision
Open in a new tab
GigaByte.

Review MS

Editor: Yunyun Lv
Reviewer name and names of any other individual's who aided in reviewer Yunyun Lv
Do you understand and agree to our policy of having open and named reviews, and having your review included with the published papers. (If no, please inform the editor that you cannot review this manuscript.) Yes
Is the language of sufficient quality? Yes
Please add additional comments on language quality to clarify if needed
Are all data available and do they match the descriptions in the paper? Yes
Additional Comments
Are the data and metadata consistent with relevant minimum information or reporting standards? See GigaDB checklists for examples <a href="http://gigadb.org/site/guide" target="_blank">http://gigadb.org/site/guide</a> Yes
Additional Comments
Is the data acquisition clear, complete and methodologically sound? Yes
Additional Comments
Is there sufficient detail in the methods and data-processing steps to allow reproduction? Yes
Additional Comments
Is there sufficient data validation and statistical analyses of data quality? No
Additional Comments Appropriate modifications
Is the validation suitable for this type of data? Yes
Additional Comments
Is there sufficient information for others to reuse this dataset or integrate it with other data? Yes
Additional Comments
Any Additional Overall Comments to the Author The main contribution of this article is a new chromosome-level genomic assembly of humpback puffer with NGS, TGS and Hi-C reads. The high-quality of the assembly is reflected in the results and figures. However, I feel some results should be checked and some sentence should be rephrased until it could be officially received. I list the lines with errors or that need to be rewritten. In summary, I feel this article could be acceptable after major revisions. Abstract: “ Despite interesting biological features, such as its very inactive nature, tetrodotoxin production and body expansion mechanisms, molecular research on the humpback puffer is still rare because of the lack of a high-quality reference genome.” I feel this sentence should be rephrased. I understand a high-quality genome resource should benefit molecular research that focused on this species. However, I don’t think the reason of rare molecular studies is due to a lack of genomic assembly. In addition, “inactive nature” should be replaced by other words as my opinion. “scaffold N50s”? “Based on the genome, ~61.5Mb (18.11%) repeat sequences were also identified, and totally 19,925 genes were annotated, 99.20% of which could be predicted with function using protein-coding function databases.” I feel this sentence should be rewritten. In addition, the inconsistence appears in the abstract and results. There are 90.1% genes could be annotated with biological function as author’s announcement in section of genome annotation. This mistake should be fixed in the revision. ‘Finally, a phylogenetic tree was constructed with single-copy gene families from ten teleost fishes.’ This sentence just tells me you have done the phylogenetic analysis, but where the result (phylogenetic status of humpback puffer according to your analysis). The humpback puffer genome will be a valuable genomic resource to illustrate possible mechanisms of tetrodotoxin synthesis and tolerance, providing clues for future detailed studies of biological toxins. I understand this genome would be valuable for further studies, but I cannot see any clues for studying biological toxins in this article. Data Description The author mentioned slight sex dimorphism occurred between male and female, however, the sampling sex used to genomic assembly is not described. ‘Different from other species of predatory pufferfish, the humpback puffer is so inactive that it only moves when the food is right in front of it’ I feel confusion in this sentence. The humpback pufferfish is not predatory, or just owes lazy behavior in food seeking process? ‘Previous studies have proved that the content of the toxicity in the humpback puffer varies greatly in different seasons, so it can be edible when its skin and internal organs are removed’ I feel the former has no logical relationship with the latter. ‘In addition to these biological characteristics, the compact genome size of humpback puffer is roughly about 385 Mb, which alongside other pufferfish species which have been used to study intron evolution, makes it an ideal model species for genetic study’ This sentence is too long that bring difficulties to read. In this study, we provided a chromosome-scale genome of an adult humpback puffer that will allow us to study features such as mechanisms of tetrodotoxin synthesis, expansion defense, body differences between males and females, and genome size. Comparative genomics analysis can help to better understand the phenotypic evolution and special gene families of the Tetraodontidae. This sentence may mislead the readers that this article should contain those analyses related to the mentioned features, but actually not. Thus, I feel it should be rephrased. Methods To be honest, I feel the section title should be “Materials, Methods and Result” as the content actually contains the three aspects. In the assembling process, the authors assembled draft assembly with only using of stLER reads but without PB data, and the PB data was only used for closing gaps? I have not used TGSgapfiller, but I think the function of this program may be limited in gap close but not expand the assembled contigs. Why do not use the PB reads in initial draft contig assembly? How to define chromosomes number (18) in 3dDNA pipeline should be described. “1,945 bps coding region” should be “1,945 bp ” As mentioned above, the inconsistence appears in this section and Abstract ‘We firstly used MUSCLE…..’ Type error. There is a redundant space between ‘to’ and ‘align’ in this sentence. The author should describe how many fossils used to correct the phylogenetic topology and which nodes be corrected. In addition, I am curious that why parameter of alpha is 0.431879.
Recommendation Major Revision
Open in a new tab
GigaByte.

Editor Decision

Editor: Scott Edmunds
GigaByte. 2021 Apr 1;2021:gigabyte17.

Major Revision

Rui Zhang
GigaByte.

Assess Revision

Editor: Scott Edmunds
GigaByte.

Re-Review MS

Editor: Yunyun Lv
Comments on revised manuscript After this revision, some errors in the MS were fixed well. However, I feel it needs to be improved and revised as some errors is still existed as following: Abstract “Based on the genome, ~61.5Mb (18.11%) repeat sequences were identified 19,925 genes were annotated, and 90.01% of these genes could be predicted with function.” Grammatical errors. Missing comma. “Finally, a phylogenetic tree of ten teleost fish species was constructed .” The result from phylogenetic analysis is still absent in the current content. This point should be improved by adding the content “Finally, a phylogenetic tree of ten teleost fish species was constructed, which suggests …”. In addition, there is a redundant space in this sentence, this kind of error should be revised throughout the whole manuscript. Please check the manuscript carefully. Methods “A Nanopore library was constructed and sequenced on the GridION platform. In total, we obtained 120 Gb (~312 X) raw stLFR data, 19 GB (~ 49X) raw Hi-C data, and 12 GB (~ 32X) raw Pacbio data” An obvious error present in between “Nanopore library” and “raw Pacbio data”. According to author’s response, the Pacbio data should be replaced by nanopore data. We used Jellyfish (v2.2.6, RRID: SCR_005491) with 58 Gb clean stLFR reads to perform the k-17mer analysis [13], estimating the humpback buffer genome size about 385 Mb. Jellyfish can count kmer amount and species easily, however, the estimation of genome size should depend on other software or script. According to Fig. S1, GenomeScope should be the source to assist the genome size estimation. However, I can not see the citation and content mentioned that. “Finally, we perfomed the chromosomal-level scaffolding using the 3D de novo assembly (3D-DNA) pipeline (v. 170123) [17], which anchored 91.2% of total sequences to 18 chromosomes, the length ranging from 11 Mb to 35 Mb” How define the number (18) of chromosomes should be explained as I have mentioned in previous. However, the reason is still absent. “Phylogenetic analyses were performed using 4,461 single-copy protein-coding genes identified by gene family analysis.” The underline format is not required. Thus, although the improvement of this manuscript is obvious, yet it could not completely satisfy me, and I suggest minor revision for the current version.
Open in a new tab
GigaByte.

Re-Review MS

Editor: Sven Winter
Comments on revised manuscript The authors amended the manuscript, yet some important concerns they did not really address. I really think the manuscript should be published but the lack of assembly quality control, missing information about RNA sequencing, and the way they conducted the phylogenetic analyses (super matrix rather than multi-locus) should be revised again before it can be published. I would like to urge the authors to have a look at these issues again, and I really do not want to be too picky but I have the feeling, that this manuscript can still be improved quite a bit.
Open in a new tab
GigaByte.

Editor Decision

Editor: Scott Edmunds
GigaByte. 2021 Apr 1;2021:gigabyte17.

Major Revision

Rui Zhang
GigaByte.

Assess Revision

Editor: Scott Edmunds
GigaByte.

Final Data Preparation

Editor: Christopher Hunter
GigaByte.

Editor Decision

Editor: Scott Edmunds
GigaByte.

Accept

Editor: Scott Edmunds
Comments to the Author Many thanks for your submission.
Open in a new tab
GigaByte.

Export to Production

Editor: Scott Edmunds

Associated Data

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

    Data Availability Statement

    We have deposited the project at CNGB Nucleotide Sequence Archive (CNSA) where the accession ID is CNP0001025. The genomic data can be obtained in GigaScience Database [52]. The sequencing data have been deposited at National Center for Biotechnology Information (NCBI) where the bioproject accession ID is PRJNA597275.

    Articles from GigaByte are provided here courtesy of Gigascience Press

    RESOURCES