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

xiaotong niu; Yakui LV; Jin Chen; Yueheng Feng; Yilin Cui; Haorong Lu; Hui Liu

doi:10.20944/preprints202401.1712.v1

Submitted:

22 January 2024

Posted:

23 January 2024

You are already at the latest version

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.

Keywords:

genetics and genomics

;

evolutionary biology

;

zoology

Subject:

Biology and Life Sciences - Animal Science, Veterinary Science and Zoology

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 2. A protocols.io collection of protocols for sequencing snake genomes [17].

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 4. Gene annotation information obtained from our T. albolabris genome. (a) KEGG enrichment. (b) GO enrichment.

Figure 5. BUSCO Assessment result of our T. albolabris genome.

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.

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.

	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.

	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.

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.

Type	Repbase TEs		TE protiens		De novo		Combined TEs
Type	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.

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.

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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