Background & Summary

The Qinghai-Tibetan Plateau (QTP) is the largest and highest plateau in the world1. The upshift of QTP has formed complex mountain systems in Southwest China and greatly reshaped the drainage at this area2. The rapid alteration of topography in the QTP might act as significant barriers for gene flow of many species, leading to population isolations and initiating allopatric divergence and speciation3. Genomes of fish species in the QTP provide valuable resources for scientists to understand the molecular mechanism of environmental adaptation. Although we have successfully obtained the reference genome of Glyptosternon maculatum4, leading to the first high-quality fish genome in Tibet-plateau, the genome information of fish species in QTP is still lacking.

The schizothoracine fishes (Schizothoracinae subfamily, Cyprinidae family, Cypriniformes order), also known as “mountain carps”, which composed of approximately 100 species in 10–13 genera5. They can be diagnosed by two lines of enlarged scales along both sides of the urogenital opening and anus6. These fishes exhibit many unique traits that adapt to the extreme environment of the QTP7. Therefore, this taxon provides an excellent opportunity for investigating high altitude adaptation of teleost fishes.

Distributed in the QTP and its surrounding areas, they are the largest and most diverse taxon of the QTP icthyofauna6. Based on morphological traits, the schizothoracine fishes can be divided into three hierarchical groups that adapt to different environments of QTP: the primitive group (including Schizothorax, Schizocypris, and Aspiorhynchus), the specialized group (including Diptychus, Gymnodiptychus, and Ptychobarbus), and the highly specialized group (including Gymnocypris, Oxygymnocypris, Chuanchia, Herzensteinia, Platypharodon, and Schizopygopsis)6. The evolution of the three groups was proposed to be associated with the upshift history of the plateau6,8. Thus, schizothoracine fishes represent an excellent model for the study of speciation caused by geographical isolation, as well as a good model for the study of adaptive evolutions of fish species in the QTP.

Another prominent feature in the evolution of schizothoracine fishes is the complex chromosome compositions, and the majority of fishes in this taxon are considered to be polyploids9. Whole genome duplication (WGD) plays a vital role in the evolutionary history of plant and animals. There are at least three rounds of whole genome duplications early in teleost diversification10,11, and these events were suggested to be causally related to the evolutionary success of teleost12,13. The polyploid nature and rapid diversification of schizothoracine fishes make them a good model for the study of polyploidy driven speciation.

Oxygymnocypris stewartii (Lloyd, 1908) (NCBI Taxon ID: 361644, Fig. 1a), a highly specialized schzothoracine fish, is a one-time spawning fish species mainly distributed in the tributaries of the middle reaches in the YarlungZangbo River across an elevation ranging from roughly 3,000 m to 4,200 m14 (Fig. 1b). O. stewartii is currently listed in the Red List by the World Conservation Union (IUCN) and identified as an endangered fish15. Therefore, it is imperative to protect and restore the population resources of the O. stewartii.

Figure 1: A picture of Oxygymnocypris stewartii.
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(a) The appearance of Oxygymnocypris stewartii; (b) Distributed localization (red triangle) of Oxygymnocypris stewartii for the genomic sequencing.

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In this report, we provide the whole genome sequence of O. stewartii through the PacBio single molecule sequencing technique (SMRT). The availability of a fully sequenced and annotated genome is essential to support basic biological studies and will be helpful to the development of further protection strategies for this endangered species. Its whole genome sequence will also provide a foundation to explore the adaptive evolutionary processes of highland fishes, supplied as a starting point to study speciation mechanisms caused by the rapid rising of the QTP.

Methods

Sample collection and sequencing

A healthy female fish captured from Gongga Country, Lhasa, Tibet (Fig. 1a,b) was used for genome sequencing. Genomic DNA was isolated using Qiagen DNA purification kit (Qiagen, Valencia, CA, USA) from the white muscular tissue as in our previous studies4.

To generate enough read data for the genome assembly, both the PacBio SEQUEL and the Illumina HiSeq 4000 platform were used for the sequencing. Long reads generated from the PacBio platform were used for genome assembly, and the short but accurate reads from the Illumina platform were analyzed for genome survey and base level correction after the assembly. For the PacBio platform, genomic sequencing libraries were constructed according to the PacBio suggested protocol and 141.1 Gb long sequencing reads were obtained from 27 SMRT cells. A total of 140.7 Gb (coverage of 74.3×) subreads were obtained after removing adaptors in polymerase reads (Table 1). The subreads N50 and average lengths were 14.2 and 9,0 kb, respectively. For the Illumina HiSeq 4000 sequencing platform, one ug genomic DNA molecules were used for sequencing library construction. DNA molecules were fragmented, end-paired and ligated to the adaptor, which was further fractionated on agarose gels and purified by PCR amplification. To improve the representativeness of reads for the O. stewartii genome, 11 paired-end sequencing libraries were constructed with insert length of 250 bp according to Illumina’s protocol (Illumina, San Diego, CA, USA). Finally, a total of 145.4 Gb (coverage of 70.8×) short sequencing reads were generated. Reads with the adaptors and a quality value lower than 20 (corresponding to a 1% error rate) were filtered out. As a result, we obtained 144.3 Gb cleaned reads for the k-mer analysis and base correction of the genome (Table 1).

Table 1 Sequencing data used for the Oxygymnocypris stewartii genome assembly.
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The individual used for the genomic sequencing was also used for the transcriptome sequencing, providing necessary gene expression data for the genome sequence annotation. Given that gene expression exhibited clear tissue-specificity, 12 tissues, including skin, eye, swim bladder, muscle, brain, gill, heart, liver, gut, ovary, fat tissue and kidney were collected for the following transcriptome sequencing. As per the similar method in our previous study4, RNA molecules were extracted using RNAiso Pure RNA Isolation Kit (Takara, Japan) for all samples, and DNase I treatment was performed to eliminate DNA contamination. After the quality assessment of the extracted RNAs using NanoVue Plus spectrophotometer (GE Healthcare, NJ, USA), RNA-seq libraries were constructed according to the protocol4 and were sequenced by Illumina HiSeq 4000 in paired-end 150 bp mode, resulting in a total of ~50 Gb transcriptome data. All genome and transcriptome sequencing data were summarized in Table 1.

De novo assembly of Oxygymnocypris stewartii genome

Genome size was estimated using Illunima sequencing data with the Kmer-based method16. As per our previous study4, we estimated the genome size of O. stewartii by the Kmer frequency distribution. Jellyfish (v2.1.3)17 was used to calculate the frequency of each Kmer from the short sequencing data (Table 2 and Fig. 2). As a result, we estimated the genome size of O. stewartii to be approximately 1,893.5 Mb.

Table 2 Statistics of 17-mer analysis for Oxygymnocypris stewartii genome.
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Figure 2: 17-mer frequency distribution in Oxygymnocypris stewartii genomes.
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The X-axis is the Kmer depth, and Y-axis represents the frequency of the Kmer for a given depth.

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The long reads generated from the PacBio SEQUEL platform were assembled into contigs using the FALCON package18 with default parameters. After the self-error correction step in the FALCON, we got 104.9 Gb (55.4x coverage) of error-corrected pre-assembly reads. The assembly of the PacBio data alone resulted in a genome of 1,898.4 Mb with a contig N50 length of 240.3 kb. The assembled genomic sequences were further polished by two rounds of polishing with Quiver19 using the PacBio long reads. After that, another round of the genome-wide base-level correction was performed with the Illumina short sequencing data by Pilon20. In the end, we obtained the final 1,849 Mb draft genome of O. stewartii with a contig N50 length of 257.1 kb (Table 3).

Table 3 The statistics of length and number for the de novo assembled genome of Oxygymnocypris stewartii.
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The completeness and the accuracy of the genome were evaluated by CEGMA, BUSCO and read mapping. The completeness of the genome assembly was assessed by the single copy orthologs (BUSCO, version 3.0)21 and CEGMA22 software. 94.2% complete and 3.6% partial of the 2,586 vertebrate BUSCO genes were identified in the final assembly. Using CEGMA22, we revealed that 95.56% of the 248 core genes were evolutionarily conserved genes identified in the genome. Both BUSCO and CEGMA confirmed the completeness of the genome assembly. The accuracy of the genome was evaluated by the Illumina short read mapping with BWA23 and the transcript alignment with BLAT24. More than 98.6% of the reads were aligned to the genome, and the insert length distribution exhibited a single peak that was consistent with the experimental design. Meanwhile, the transcriptome was de novo assembled by Trinity25, and the transcripts were mapped to the genome assembly using BLAT24 with default parameters. We found that the alignment coverage (alignment length to transcript length) of expressed genes ranged from 96.44 to 99.95% in the genome assembly.

Repetitive element and non-coding gene annotation in the O. stewartii genome

To annotate repeat elements in the O. stewartii genome, both homologous comparison and ab initio prediction were applied. The similar annotation process in our previous work4 was employed. For ab initio repeat annotation, LTR_FINDER26, RepeatScout27, and RepeatModeler (http://repeatmasker.org/RepeatModeler/) were used to construct a de novo repetitive element database, and the RepeatMasker28 (http://repeatmasker.org/RMDownload.html) were used to annotate repeat elements with the database. Then, RepeatMasker and RepeatProteinMask28 were used for known repeat element types by searching against Repbase database29. Tandem repeats were also ab initio predicted using TRF tool30. A total of 822.84 Mb repetitive elements were identified in the O. stewartii genome by those repeat annotation processes, accounting for 44.50% of the whole genome (Tables 4 and 5 and Fig. 3).

Table 4 The annotation of repeated sequences in the Oxygymnocypris stewartii genome using TRF, RepeatMasker, and RepeatProteinMask.
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Table 5 Summary statistics of repeat annotation in Oxygymnocypris stewartii.
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Figure 3: Distribution of the divergence rate of each type of repetitive element in Oxygymnocypris stewartii genome.
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The divergence rate was calculated between the identified TE elements in the genome by the homology-based method and the consensus sequence in the Repbase.

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For non-coding genes, 24,208 tRNAs were predicted using tRNAscan-SE31, and 1,363 rRNA genes were annotated using BLASTN tool with an E-value of 1E-1032 against human rRNA sequence. Small nuclear and nucleolar RNAs in the O. stewartii genome were also annotated by the infernal tool33 using Rfam database34 (Table 6).

Table 6 The number of the annotated non-coding RNA in the Oxygymnocypris stewartii genome.
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Protein-coding gene prediction and functional annotation

The gene model prediction method in our previous study4 was applied to the protein-coding gene annotation in the O. stewartii genome. We merged the evidence of the gene prediction from multiple methods, including homolog based, ab initio and RNA-seq based annotations. The protein and coding sequences were obtained from the Ensembl database35 for the following species, including human (Homo sapiens, GCF_000001405.37), mouse (Mus musculus, GCF_000001635.26), zebrafish (Barchydanio rerio var, GCF_000002035.5), common carp (Cyprinus carpio, GCF_000951615.1), tiger puffer (Takifugu rubripes, GCF_000180615.1), channel catfish (Ictalurus punctatus, GCF_001660625.1), Sinocyclocheilus graham (GCF_001515645.1) and grass carp36 (Ctenopharyngodon idellus). The protein sequences were aligned against the O. stewartii genome using TBLASTN37 search with parameters of e-value 1e-5. After filtering low-quality records, the gene structure was predicted by GeneWise38 (referred to “Homology” in Table 7). Secondly, transcripts assembled from twelve tissues RNA-Seq data were aligned against the O. stewartii genome using Program to Assemble Spliced Alignment (PASA)39 (referred to “PASA” in Table 7). Augustus40, GeneID41, GeneScan42, GlimmerHMM43, and SNAP44 were used for ab initio prediction with the optimized parameters that trained using high-quality proteins that derived from the PASA gene models. RNA-seq reads were also aligned to the O. stewartii genome directly using TopHat45 v2.0.9, and the gene models were constructed by Cufflinks46 v2.2.1 (referred to Cufflinks in Table 7). Finally, EvidenceModeler39 was applied to combine all gene models that were predicted by various methods with the identical weights with our previous work4. Untranslated regions (UTRs) and alternative splicing variations were annotated using PASA239 (referred to “PASA-update” in Table 7). Finally, 46,400 protein-coding genes with a mean of 8.41 exons per gene (Table 7) were annotated in the O. stewartii genome. The statistics of gene models, including lengths of a gene, CDS, intron, and exon in O. stewartii were comparable to those for close-related species (Table 8 and Fig. 4).

Table 7 The statistics of gene models of protein-coding genes annotated in the Oxygymnocypris stewartii genome.
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Table 8 The comparison of the gene models annotated from the Oxygymnocypris stewartii genome and other teleosts.
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Figure 4: Comparisons of the prediction gene models in the Oxygymnocypris stewartii genome to other species.
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(a) CDS length distribution and comparison with other species. (b) Exon length distribution and comparison with other species. (c) Exon number distribution and comparison with other species. (d) Gene length distribution and comparison with other species. (e) Intron length distribution and comparison with other species.

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Public biological function databases of SwissProt47, InterPro48, NR from NCBI and Kyoto Encyclopedia of Genes and Genomes (KEGG)49 were used for the functional annotation of the predicted genes. BLASTX utility32 were used for the homolog search with an E-value threshold of 1E-5. InterPro database48 was used to predict protein function based on the conserved protein domains by InterproScan tool50. A total of 45,991 genes (99.1%) were successfully annotated by at least one public database. (Table 9 and Fig. 5).

Table 9 The number of genes with homology or functional classification for Oxygymnocypris stewartii.
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Figure 5
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Venn diagram of the number of genes with functional annotation using multiple public databases.

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Code Availability

The sequence data were generated using the software provided by the sequencing platform manufacturer and the sequencing data were processed with commands with the guidance from the public software that is cited in the manuscript. No custom computer codes were generated in this work.

Data Records

All PacBio long-read sequencing data and Illumina short-read sequencing data have been deposited to NCBI Sequence Read Archive (SRA) (Data Citation 1).

The transcriptome data are available through the NCBI SRA (Data Citation 2).

The assembled genome version is available at GenBank (Data Citation 3).

The annotation gff3 file of the assembled genome is available at Figshare (Data Citation 4).

Technical Validation

RNA integrity

The transcriptomes for twelve tissues from three fish individuals were sequenced. Before constructing RNA-Seq libraries, the concentration and quality of total RNA were evaluated using NanoVue Plus spectrophotometer (GE Healthcare, NJ, USA). The total amount of RNA, RNA integrity and rRNA ratio were used to estimate the quality, content and degradation level of RNA samples. In the present study, RNAs samples with a total RNA amount ≥10 μg, RNA integrity number ≥8, and rRNA ratio ≥1.5 were finally subjected to construct the sequencing library.

Quality filtering of Illumina sequencing raw reads

The raw sequencing reads generated from the Illumina platform were rigorously cleaned by the following procedures as in the previous study4. Firstly, adaptors in the reads were filtered out; secondly, reads with more than 10% of N bases were filtered out; thirdly, reads with more than 50% of the low-quality bases (phred quality score <= 5) were filtered out. If any end pair was classified as low quality, both pairs were discarded. The initially generated raw sequencing reads were also evaluated for quality distribution, GC content distribution, base composition, average quality score at each position and other metrics.

Additional information

How to cite this article: Liu, H. P. et al. The sequence and de novo assembly of Oxygymnocypris stewartii genome. Sci. Data. 6:190009 https://doi.org/10.1038/sdata.2019.9 (2019).

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