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The Genome Assembly and Annotation of the White‐ Lipped Tree Pit Viper Trimeresurus albolabris

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22 January 2024

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23 January 2024

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Abstract
Trimeresurus albolabris, also known as the white-lipped pit viper or white-lipped tree viper, is a highly venomous snake distributed across Southeast Asia and the cause of many snakebite cases. In this study, we report the first whole genome assembly of T. albolabris obtained with next-generation sequencing from a specimen collected in Mengzi, Yunnan, China. After genome sequencing and assembly, the genome of this male T. albolabris individual was 1.51 Gb in length and included 38.42% repeat-element content. Using this genome, 21,695 genes were identified, and 99.17% of genes could be annotated using gene functional databases. Our genome assembly and annotation process was validated using a phylogenetic tree, which included six species and focused on single-copy genes of nuclear genomes. This research will contribute to future studies on Trimeresurus biology and the genetic basis of snake venom.
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Introduction

Trimeresurus albolabris, also known as the white-lipped pit viper, white-lipped tree viper, white-lipped bamboo pit viper, and green tree pit viper, is a venomous snake species belonging to the family Viperidae [1]. It is a relatively small snake, with adults typically measuring around 70-90 cm in length, and is known for its distinctive appearance, with a white stripe running down the center of its upper lip [2] (Figure 1). This species has been reported in China, Vietnam, Thailand, Laos, Cambodia, India, Bangladesh, Myanmar, and West Java and has become one of the most common venomous snakes with medical importance in Southeast Asia [3]. T. albolabris is a highly venomous snake. Its bite can be dangerous to humans, causing symptoms ranging from pain and swelling to more severe ones, such as shock, spontaneous bleeding, defibrination, and other complications of thrombocytopenia and leukocytosis [4,5]. Notably, the venom of T. albolabris contains metalloproteinases [6,7], a thrombin-like enzyme [8], and other venom components [5,9].

Context

Despite its venomous nature, T. albolabris is also an important research subject for its sexual dimorphism [10] and geographic variation [11]. A complete and high-quality genome of this species is crucial for studying venom proteomics, particularly for drug discovery, developing antivenom therapies, and understanding the evolution of venomous species [12,13,14]. However, a complete genome of T. albolabris has not been published yet [15].
Here, we report the first whole genome with high continuity of a male T. albolabris individual, collected from Mengzi, Yunnan, China. The genome was generated using single-tube long fragment read (stLFR) [16] and whole genome sequencing (WGS) technologies. Our T. albolabris genome had a repeat element content of 38.42% and a total size of 1.51 Gb. This new genome assembly provides valuable evidence for future studies on snake venom and the genetic underpinnings of the Trimeresurus species.

Method

The comprehensive and sequential procedures employed in this investigation are compiled within a protocols.io compilation, with the slight adjustments delineated underneath (Figure 2). [17].

Sample Collection and Sequencing

In Mengzi, Yunnan, China, we captured a male T. albolabris specimen. To ensure its preservation, the specimen was promptly frozen in dry ice (at -80°C) right after collection and identification, serving both for storage and transportation purposes. Detailed procedures for DNA extraction, library construction, and sequencing are available in a protocols.io protocol collection [17]. For RNA sequencing, we utilized the heart, stomach, liver, and kidneys, while a muscle sample was employed for stLFR and WGS sequencing. The genome assembly and annotation workflow can also be found in the corresponding protocols.io protocol [17].
Approval for this research, encompassing sample gathering, experimental processes, and study design, was granted by the Institutional Review Board of Beijing Genomics Institute (BGI-IRB E22017). Throughout the investigation, scrupulous adherence to the protocols outlined by BGI-IRB was rigorously maintained, ensuring alignment with ethical and regulatory norms.

Genome Assembly, Annotation, and Assessment

The stLFR sequencing data were subjected to assembly using Supernova [18] (v2.1.1, RRID:SCR_016756). Subsequently, the gap-filling and redundancy removal steps were performed using GapCloser [19] (v1.12-r6, RRID:SCR_015026) and redundans [20] (v0.14a), respectively. These processes involved utilizing the WGS data to address the gaps in the assembly and eliminate redundant sequences.
To identify repeat elements within the genome sequences, multiple genomic tools were utilized, including Tandem Repeats Finder [21] (v. 4.09, RRID:SCR_022193), LTR_Finder [22] (RRID:SCR_015247), RepeatModeler [23] (v1.0.8), RepeatMasker [24] (v. 3.3.0, RRID:SCR_015027), and RepeatProteinMask (v. 3.3.0) [25]. Predicting protein-coding genes involved a comprehensive strategy combining de novo, homology-based, and transcript mapping approaches. De novo gene prediction was performed using GlimmerHMM [26] (RRID:SCR_002654). RNA-seq-based predictions began with Trimmomatic [27] (v0.30, RRID:SCR_011848) filtering of RNA-seq data. Following the acquisition of clean RNA-seq data, Trinity [28] (v2.13.2, RRID:SCR_013048). Finally, PASA [29] (v2.0.2, RRID:SCR_014656) aligned transcripts against the white-lipped tree viper genome to derive gene structures. Homology-based prediction involved mapping protein sequences from the UniProt database (release-2020_05), including Pseudonaja textilis (GCA_900518735.1), Protobothrops mucrosquamatus (GCA_001527695.3), Thamnophis elegans (GCA_009769535.1), and Notechis scutatus (GCA_900518725.1) to the white-lipped tree viper genome using Blastall (v2.2.26) [30] with an E-value cut-off of 1e-5. Then, we used GeneWise [31] (v2.4.1, RRID:SCR_015054) to analyze the alignment results and predict gene homology. The integration of RNA-seq, homology, and de novo predicted genes resulted in the generation of a final gene set using the MAKER pipeline (v3.01.03, RRID:SCR_005309) [32]. This approach, incorporating multiple genomic tools and techniques, facilitated the annotation and prediction of genes in the white-lipped tree viper genome.
The functional characterization was conducted through a BLAST investigation, contrasting with various repositories such as SwissProt, TrEMBL, and Kyoto Encyclopedia of Genes and Genomes (KEGG), while restricting the E-value threshold to 1e-5. InterProScan [26] (v5.52-86.0, RRID:SCR_005829) was employed to anticipate patterns and domains, along with gene ontology (GO) descriptors.
Assessing the integrity of our genome involved the utilization of Benchmarking Universal Single-Copy Orthologs (BUSCO, v5.2.2, RRID:SCR_015008) in genome mode, employing lineage information from vertebrata_odb10 for benchmarking purposes [33].
A reconstructed phylogenetic tree was generated by OrthoFinder (v2.3.7, RRID:SCR_017118) [34], which can search for single-copy orthologs among the protein sequences of Chelonia mydas (GCA_015237465.2), Gallus gallus (GCA_016699485.1), Homo sapiens (GCA_000001405.29), Mus musculus (GCA_000001635.9), Ophiophagus hannah (GCA_000516915.1), Python bivittatus (GCA_000186305.2), Xenopus tropicalis (GCA_000004195.4), Alligator mississippiensis (GCA_000281125.4), Danio rerio (GCA_000002035.4), Anolis carolinensis (GCA_000090745.2), Gopherus evgoodei (GCA_007399415.1), Podarcis muralis (GCA_004329235.1), and Deinagkistrodon acutus [35].

Results

This study on snake genomics resulted in a total of 387.48 Gb of paired-end (fastq 1 and fastq 2) data, which comprised 204.61 Gb of short reads data obtained through WGS sequencing and 182.87 Gb of long reads data obtained through stLFR sequencing, as shown in Table 1 and Table 2.
We produced the first whole of the genomic structure for T. albolabris, demonstrating remarkable continuity, encompassing a comprehensive genome size of 1.51 Gb, a GC content of 39.97%, and an N50 scaffold length of 381.55 kb (Table 3). The compiled genome of T. albolabris comprises 10,016 contigs surpassing 1,000 base pairs, with a collective extension of 1.50 Gb, representing 99.14% of the overall genome length. This asset will offer substantial proof for delving into novel perspectives in the examination of Trimeresurus viper genomics.
We detected repetitive elements in the T. albolabris genome, accounting for 38.42% of the total genome. Among them, the highest proportion was occupied by long interspersed nuclear elements (LINEs), which accounted for 23.94% and amounted to approximately 362.35 Mb. These findings were found to be highly similar to the repetitive element content observed in previously sequenced genomes, such as those of Thamnophis elegans (42.02%) (accession No. PRJNA561996) and Crotalus tigris (42.31%) [36]. This indicates that the results we obtained are highly reliable and plausible. The remaining types of transposable elements, including DNA transposons, long terminal repeats (LTRs), and short interspersed nuclear elements (SINEs), accounted for 6.90%, 5.83%, and 1.24%, respectively (Figure 3, Table 4, and Table 5).
Using homology-based, de novo, and RNA-sequencing annotation methods, we successfully identified 21,695 protein-coding genes in our T. albolabris genome assembly. We compared our assembly to those of Notechis scutatus (GCA_900518725.1), Pseudonaja textilis (GCA_900518735.1), and Thamnophis elegans (GCA_009769535.1), all of which are available from the NCBI database. Our analysis revealed no significant differences in the distribution of transcript mapping lengths, coding sequences (CDS) lengths, or the quantity of exons and introns. Additionally, our analysis predicted the presence of 250 miRNAs, 179 tRNAs, and 301 snRNAs within the T. albolabris genome (Table 6).
Comparing our results with various public datasets, such as InterPro [37], KEGG [38], SwissProt [39], TrEMBL [39], and GO terms, we identified 21,695 expanded gene families, including 99.17% functionally annotated genes (Table 7).
Further analyses using KEGG enrichment revealed that Environmental Information Processing, Organismal Systems, and Metabolism pathways were the most abundant, with Signal Transduction pathways being the most prominent. Among the Organismal Systems pathways, 1,774 Immune System genes and 1,551 Endocrine System genes were the most abundant (Figure 4a). In addition, based on the results of our GO analysis, we found that 7,900 genes are related to binding, while 7,740 genes are related to cellular processes (Figure 4b).

Data Validation and Quality Control

We employed BUSCO v5.2.2 to assess the quality and completeness of our genome assembly [40]. The results of our BUSCO analysis revealed that our assembly achieved 85.3% completeness when evaluated against the vertebrata_odb10 database (Figure 5), indicating that our assembly is of relatively high quality and completeness.
To evaluate the integrity of our assembly, we generated a phylogenetic tree using the protein sequences of seven distinct amphibian and reptile species (Anolis carolinensis, Chelonia mydas, Deinagkistrodon acutus, Ophiophagus hannah, Python bivittatus, Xenopus tropicalis, and Alligator mississippiensis), along with the protein sequences of Gallus gallus, Homo sapiens, Mus musculus, and Danio rerio obtained from NCBI. The resultant phylogenetic tree aligns with prior research findings, affirming that our data can reliably discern relationships among species (Figure 6).

Reuse Potential

We unveiled the inaugural genome assembly of the white-lipped arboreal pit viper. This information furnishes novel tools for delving into the serpent's biological and evolutionary aspects, along with the genetic underpinnings of its venom.

Author Contributions

H Liu and H Lu designed and initiated the project. Y C and Y F performed the DNA extraction and the library construction. X N and J C performed the data analysis. X N, J C and Y L wrote the manuscript. All authors read and approved the final manuscript.

Data Availability Statement

The information substantiating the conclusions of this research has been stored in the CNGB Sequence Archive (CNSA) [41] within the China National GeneBank DataBase (CNGBdb) [42] , under the designated accession number CNP0004151. The unprocessed data can be accessed on NCBI using the Bioproject number PRJNA955401 (see also the machine readable nanopublication: RAV3oIcruk). Supplementary data is also accessible through GigaDB [43].

Editor’s note

This article is a component of a collection of Data Release manuscripts introducing the genetic codes of diverse serpent varieties [44].

Ethics approval

The research conducted in this study received approval from the Institutional Review Board of BGI (BGI-IRB E22017).

Acknowledgments

Our initiative received backing from the China National GeneBank (CNGB). Additionally, Hainan University and BGI-Shenzhen provided support for this endeavor. The sample collection was carried out by Anhui Normal University. Funding for this project was granted by the Guangdong Provincial Key Laboratory of Genome Read and Write (grant no. 2017B030301011).

Competing Interests

The authors declare no competing interests.

Abbrevitions

fAbbreviations used include fq for fastq, GO for gene ontology, KEGG for Kyoto Encyclopedia of Genes and Genomes, LINE for long interspersed nuclear element, LTR for long terminal repeat, SINE for short interspersed nuclear element, stLFR for single-tube long fragment read, TEs for transposable elements, and WGS for whole genome sequencing.

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Figure 1. Trimeresurus albolabris, also known as the white-lipped pit viper, photographed by Diancheng Yang.
Figure 1. Trimeresurus albolabris, also known as the white-lipped pit viper, photographed by Diancheng Yang.
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Figure 2. A protocols.io collection of protocols for sequencing snake genomes [17].
Figure 2. A protocols.io collection of protocols for sequencing snake genomes [17].
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Figure 3. Distribution of transposable elements (TEs) in our T. albolabris genome. These TEs encompass DNA transposons (referred to as DNA) and RNA transposons (specifically, DNAs, LINEs, LTRs, and SINEs). (a) Distribution of the de novo sequence divergence-rate. (b) Distribution of the known sequence divergence-rate.
Figure 3. Distribution of transposable elements (TEs) in our T. albolabris genome. These TEs encompass DNA transposons (referred to as DNA) and RNA transposons (specifically, DNAs, LINEs, LTRs, and SINEs). (a) Distribution of the de novo sequence divergence-rate. (b) Distribution of the known sequence divergence-rate.
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Figure 4. Gene annotation information obtained from our T. albolabris genome. (a) KEGG enrichment. (b) GO enrichment.
Figure 4. Gene annotation information obtained from our T. albolabris genome. (a) KEGG enrichment. (b) GO enrichment.
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Figure 5. BUSCO Assessment result of our T. albolabris genome.
Figure 5. BUSCO Assessment result of our T. albolabris genome.
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Figure 6. Phylogenetic tree reconstructed employing single-copy genes from the nuclear genome. The numerical values associated with the branches in the phylogenetic tree indicate the branch distances determined through the utilization of OrthoFinder.
Figure 6. Phylogenetic tree reconstructed employing single-copy genes from the nuclear genome. The numerical values associated with the branches in the phylogenetic tree indicate the branch distances determined through the utilization of OrthoFinder.
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Table 1. Summary statistics of T. albolabris of WGS paired-end (fq- fastq 1 and fastq 2) sequenced reads.
Table 1. Summary statistics of T. albolabris of WGS paired-end (fq- fastq 1 and fastq 2) sequenced reads.
WGS-1 WGS-2 WGS-3
fq1 fq2 fq1 fq2 fq1 fq2
%Q20 96.98 97.81 97.74 94.96 95.69 97.59
%Q30 90.79 90.6 92.83 84.27 84.46 89.81
%GC 40.37 40.21 41.02 40.81 40.36 40.47
%ErrorRate 0.351809 0.233019 0.264481 0.540272 0.449364 0.257064
TotalReads 492,445,828 425,689,572 104,911,172
TotalBases 98,489,165,600 85,137,914,400 20,982,234,400
Table 2. Summary statistics of T. albolabris stLFR and RNA sequenced reads.
Table 2. Summary statistics of T. albolabris stLFR and RNA sequenced reads.
stLFR-1 stLFR-2 RNA-seq
fq1 fq2 fq1 fq2 fq1 fq2
%Q20 96.53 95.59 96.41 96.3 98.3 98.19
%Q30 89.83 87.26 87.98 86.37 94.3 93.71
%GC 39.34 42.16 39.28 42.15 44.11 44.07
%ErrorRate 0.403065 0.525486 0.442228 0.392415 0.194523 0.205665
TotalReads 633,976,833 161,105,172 50,828,075
TotalBases 145,814,671,590 37,054,189,560 10,165,615,000
Table 3. Summary of the features of the T. albolabris genome.
Table 3. Summary of the features of the T. albolabris genome.
contigs contigs >(1,000bp) contigs >(10,000bp)
Total number (>) 71,131 46608 10,016
Total length of (bp) 1,513,852,334 1,501,212,553 1,355,102,082
N50 Length (bp) 381,553
N75 Length (bp) 115,212
GC content is (%) 39.97
Table 4. Statistics of the repetitive sequences identified in our T. albolabris genome.
Table 4. Statistics of the repetitive sequences identified in our T. albolabris genome.
Type Repeat Size % of genome
Trf 47,767,541 3.155363
Repeatmasker 252,985,952 16.711402
Proteinmask 185,792,360 12.272819
De novo 498,353,737 32.919574
Total 581,568,803 38.416482
Table 5. Summary of the TEs in our T. albolabris genome.
Table 5. Summary of the TEs in our T. albolabris genome.
Type Repbase TEs TE protiens De novo Combined TEs
Length (Bp) % in genome Length (Bp) % in genome Length (Bp) % in genome Length (Bp) % in genome
DNA 51,357,881 3.392529 2,032,636 0.134269 64,605,374 4.267614 104,513,127 6.903786
LINE 184,866,441 12.211656 157,414,659 10.398284 294,829,469 19.475444 362,351,919 23.935751
SINE 9,622,825 0.635651 0 0 13,144,889 0.868307 18,769,499 1.23985
LTR 23,685,560 1.564589 26,413,572 1.744792 74,868,256 4.945546 88,305,953 5.833195
Other 77,658 0.00513 141 0.000009 0 0 77,799 0.005139
Unknown 0 0 0 0 98,895,691 6.532717 98,895,691 6.532717
Total 252,985,952 16.711402 185,792,360 12.272819 496,342,637 32.786727 566,552,754 37.424572
Table 6. Statistics for the miRNA, tRNA, rRNA, and snRNA discerned from our T. albolabris genome.
Table 6. Statistics for the miRNA, tRNA, rRNA, and snRNA discerned from our T. albolabris genome.
Type Copy(w) Average length(bp) Total length(bp) % of genome
miRNA 250 98.992 24,748 0.001635
tRNA 179 75.70949721 13,552 0.000895
rRNA 104 137.6057692 14,311 0.000945
snRNA 301 115.1229236 34,652 0.002289
Table 7. Consequences of gene functional annotation.
Table 7. Consequences of gene functional annotation.
Values Total Swissprot-Annotated KEGG-Annotated TrEMBL-Annotated Interpro-Annotated GO-Annotated Overall
Number 21,695 20,240 19,216 21,134 21,019 14,786 21,516
Percentage 100% 93.29% 88.57% 97.41% 96.88% 68.15% 99.17%
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