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https://doi.org/10.20944/preprints202306.0658.v1
Data Release
Genetics and Genomics
Evolutionary Biology
Zoology
The genome assembly and annotation of the many-banded krait, Bungarus multicinctus
B. Liu et al.
The genome assembly and annotation of the many-banded krait, Bungarus multicinctus
https://orcid.org/0000-0001-7378-3454
Liu Boyang 1 Formal analysis †
Cui Liangyu 1 Writing - original draft †
Deng Zhangwen 2 Resources †
Ma Yue 1 Methodology
Yang Diancheng 3 4 Methodology
Gong Yanan 3 4 Methodology
Xu Yanchun 1 Writing - review editing
Lan Tianming 5 6 Methodology
Yang Shuhui 1 Writing - review editing *
https://orcid.org/0000-0001-6786-8523
Huang Song 3 4 Project administration *
1 College of Wildlife and Protected Area, Northeast Forestry University, Harbin 150040, China
2 Guangxi Forest Inventory and Planning Institute, Nanning 530011, China
3 Anhui Province Key Laboratory of the Conservation and Exploitation of Biological Resource, College of Life Sciences, Anhui Normal University, Wuhu 241000, China
4 Huangshan Noah Biodiversity Institute, Huangshan 245000, China
5 BGI Life Science Joint Research Center, Northeast Forestry University, Harbin 150040, China
6 State Key Laboratory of Agricultural Genomics, BGI-Shenzhen, Shenzhen 518083, China
* Corresponding authors. E-mail: snakeman@ahnu.edu.cn; yangshuhui@nefu.edu.cn
† Contributed equally.
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2023
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© The Author(s) 2023.
2023
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Snakes are a vital component of wildlife resources and are widely distributed across the globe. The many-banded krait Bungarus multicinctus is a highly venomous snake found across Southern Asia and central and southern China. Snakes are an ancient reptile group, and their genomes can provide important clues for understanding the evolutionary history of reptiles. Additionally, genomic resources play a crucial role in comprehending the evolution of all species. However, snake genomic resources are still scarce. Here, we present a highly contiguous genome of B. multicinctus with a size of 1.51 Gb. The genome contains a repeat content of 40.15%, with a total length exceeding 620 Mb. Additionally, we annotated a total of 24,869 functional genes. This research is of great significance for comprehending the evolution of B. multicinctus and provides genomic information on the genes involved in venom gland functions.
Fundamental Research Funds for the Central Universities 2572020DY02 Guangdong Provincial Key Laboratory of Genome Read and Write 2017B030301011 China National GeneBank (CNGB) This work was supported by the Fundamental Research Funds for the Central Universities (No. 2572020DY02) and the Guangdong Provincial Key Laboratory of Genome Read and Write (grant No. 2017B030301011). This work was also supported by China National GeneBank (CNGB).
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pmcIntroduction
Snakes are a fascinating group of reptiles that exhibit unique and diverse characteristics. With approximately 3,070 extant species in all continents except Antarctica [1], they are known for lacking limbs, elongated body shapes, and an exclusively carnivorous diet. Snakes have evolved many specialized adaptations, such as infrared sensing pits and a venom apparatus, which provide them with exceptional predatory capabilities [1]. These adaptations have made snakes important model organisms for evolutionary studies, yielding insights into limb development, sex chromosome evolution, and venom evolution. In recent years, genetic approaches have become increasingly important in understanding the evolution and diversity of snakes [2]. By exploring the evolution of venomous snakes, we can gain a deeper understanding of the ecological and evolutionary roles of these intriguing species.
Bungarus multicinctus (NCBI:txid8616), also known as the many-banded krait or umbrella snake, is widely distributed throughout southern Asia, its range spanning across countries such as India, Pakistan, Indonesia, Sri Lanka, Malaysia, Bangladesh, Vietnam, and China [3]. B. multicinctus is recognized as one of the ten most venomous snakes in China, with a lethality rate ranging from 26.9% to 33.3% [4].
In this study, we collected a muscle sample of B. multicinctus to generate a highly contiguous genome with a genome size of 1.51 Gb. Its repeat element content reached 41.68%, providing new evidence for understanding the relationship between repeat elements and genome size in Elapidae species.
Main content
Context
This study presents a highly continuous genome assembly of B. multicinctus. The genome size of B. multicinctus was found to be 1.51 Gb, with a GC content of 37.8% (Table 1). The maximal scaffold length was 39.68 Mb, and the N50 length was 6.55 Mb, indicating a highly continuous genome sequence. This draft genome sequence of B. multicinctus will serve as an invaluable resource for further research on venomous snakes, enabling a better understanding of their genetic makeup.
Table 1 Summary of the features of our B. multicinctus genome.
Contig Scaffold
Maximal length (bp) 468,983 41,606,426
N90 (bp) 5,806 30,083
N50 (bp) 33,081 6,870,761
Number ≥ 100bp 163,090 82,383
Number ≥ 2kb 81,775 22,350
Ratio of Ns 0.045 0.045
GC content (%) 39.6 37.8
Genome size (bp) 1,548,488,562 1,621,955,402
The content of repetitive elements in our B. multicinctus genome was surprisingly large, reaching 41.68% with a total length of 675 Mb (Table 2). We analyzed the content of various repeating elements. While unknown types accounted for 51% of the repeating elements, LINEs and DNA transposons accounted for 10% and 8%, respectively (Figure 1). Research indicates that although snake species have similar genome sizes, they exhibit significant differences in TE content, with low diversity in the types of TEs [5]. Specifically, species with a longer evolutionary history tend to have higher TE diversity [6]. Our results suggest that the significant expansion of repeating elements is an important manifestation of species differences.
Figure 1. Distribution of transposable elements (TEs) in the B. multicinctus genome. The TEs include DNA transposons (DNA) and RNA transposons (i.e., DNAs, LINEs (Long Interspersed Nuclear Elements), LTRs (Long Terminal Repeats), and SINEs (Short interspersed nuclear elements)). (a) Distribution of the de novo sequence divergence rate. (b) Distribution of the known sequence divergence rate. (c) Proportion and distribution of repeating elements.
Table 2 Summary of TEs in our B. multicinctus genome.
Repbase TEs TE proteins De novo Combined TEs
Type Length (bp) % in genome Length (bp) % in genome Length (bp) % in genome Length (bp) % in genome
DNA 32,816,331 2.02 2,921,569 0.18 112,067,211 6.91 129,267,220 7.97
LINE 174,481,405 10.76 154,961,354 9.60 276,722,230 17.07 301,624,987 18.61
SINE 13,524,698 0.83 0 0 39,754,823 2.45 43,837,124 2.70
LTR 23,313,679 1.44 30,431,704 1.88 52,496,522 3.24 60,898,786 3.76
Other 16,171 0.01 243 0.01 0 0 16,414 0.01
Unknown 0 0 0 0 182,574,604 11.26 182,574,604 11.26
Total 234,804,260 14.49 188,249,038 11.61 645,464,460 39.82 675,577,436 41.68
We identified 24,869 functional genes of B. multicinctus and annotated them with Kyoto Encyclopedia of Genes and Genomes (KEGG). The majority of these genes were found to be involved in pathways related to Environmental Information Processing and Metabolism. This suggests that signal transduction-related genes play an important role in B. multicinctus (Figure 2). In addition, B. multicinctus genes were enriched in twelve metabolic pathways. The most enriched one was Lipid metabolism, and the least enriched one was Biosynthesis of other secondary metabolites.
Figure 2. Gene annotation information of B. multicinctus. (a) KEGG enrichment of B. multicinctus. (b) Gene Ontology (GO) enrichment of B. multicinctus.
Data validation and quality control
We conducted a BUSCO (v5.2.2) (RRID:SCR_015008) assessment on the assembly to evaluate its integrity [7]. The assembly captured 90.9% of complete BUSCOs in the vertebrata_odb10 dataset (Figure 3).
Figure 3. BUSCO Assessment result of our B. multicinctus genome.
To construct a phylogenetic tree, we screened closely related species, including Anolis carolinensis, Chelonia mydas, Danio rerio, Deinagkistrodon acutus, Gallus gallus, Homo sapiens, Mus musculus, Ophiophagus hannah, Python bivittatus, Xenopus tropicalis, and Alligator mississippiensis. Our data is consistent with previous studies and can be used to construct a phylogenetic tree that clusters closely related species (Figure 4) [8].
Figure 4. Phylogenetic tree reconstructed using nuclear genome single-copy genes.
Methods
Detailed stepwise protocols are gathered in a protocols.io collection (Figure 5) [9] and summarised here.
Figure 5. Protocols.io protocol collection for snake genome assembly [9]. https://www.protocols.io/widgets/doi?uri=dx.doi.org/10.17504/protocols.io.5jyl8j6e9g2w/v2
Sample collection and sequencing
B. multicinctus specimens were collected from Beiliu Longgukeng, Guangxi, and immediately transferred to dry ice for quick freezing. The samples were then stored at −80 °C. High-molecular-weight DNA was isolated using the protocol described by Wang et al. [10], and an stLFR co-barcoding DNA library was constructed using the MGIEasy stLFR Library Prep Kit (MGI, China). The libraries were sequenced using a BGISEQ-500 sequencer (RRID:SCR_017979) [11]. In addition, genomic DNA was isolated using the AxyPrep genomic DNA kit (AxyPrep, USA) for whole-genome sequencing.
We extracted the total RNA using the TRlzol reagent (Invitrogen, USA) following the manufacturer’s protocol. RNA quality, purity, and quantity were assessed using the Qubit 3.0 fluorometer (Life Technologies, USA) and the Agilent 2100 Bioanalyzer System (Agilent, USA). The cDNA libraries were generated by reverse-transcribing RNA fragments of 200–400 bp. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Northeast Forestry University.
Genome assembly, annotation and assessment
The stLFR sequencing data obtained from the many-banded krait were subjected to assembly using Supernova (v2.1.1, RRID:SCR_016756) [12]. To improve the quality of the assembly, GapCloser (v1.12-r6, RRID:SCR_015026) and redundans (v0.14a) [9] were utilized for gap filling and redundancy removal, respectively, by incorporating the whole genome sequencing data.
To identify known repeat elements in the genome of the many-banded krait, Tandem Repeats Finder [13], LTR_FINDER (RRID:SCR_015247) [11], and RepeatModeler (v2.0.1, RRID:SCR_015027) [14] were utilized. RepeatMasker (v3.3.0, RRID:SCR_012954) [15] and RepeatProteinMask v3.3.0 [16] were employed for repeat element annotation. Protein-coding genes were predicted using de novo, homology-based, and transcript-mapping approaches. The de novo gene prediction was performed using Augustus (v3.0.3, RRID:SCR_008417) [17]. RNA-seq data were filtered using Trimmomatic (v0.30, RRID:SCR_011848) [18], and transcripts were assembled based on clean RNA-seq data using Trinity (v2.13.2, RRID:SCR_013048) [19] for RNA-seq-based prediction. PASA v2.0.2 [20] was utilized to align transcripts against the many-banded krait genome to obtain gene structures. Our homology-based prediction was performed by mapping protein sequences of the UniProt database (release-2020_05) of Pseudonaja textilis, Crotalus tigris, Thamnophis elegans, and Notechis scutatus to the B. multicinctus genome using Blastall v2.2.26 [21]. Gene models were predicted by analyzing the alignment results using GeneWise (v2.4.1, RRID:SCR_015054) [22]. Finally, the MAKER pipeline (v3.01.03, RRID:SCR_005309) [23] was employed to generate the final gene set, which represented RNA-seq, homology, and de novo predicted genes.
To perform functional annotations, a BLAST search (RRID:SCR_004870) was conducted against several databases, including SwissProt [24], TrEMBL [24], and KEGG [25], with an E-value cut-off of 1e-5. Furthermore, InterProScan (v5.52-86.0, RRID:SCR_005829) [26] was used to predict motifs, domains, and GO terms.
The genome completeness was evaluated by analyzing sets of BUSCO v5.2.2 using genome mode and lineage data from vertebrata_odb10 [27], following the standard scientific methodology.
To reconstruct the phylogenetic tree, OrthoFinder (v2.3.7, RRID: SCR_017118) [28] was used to search for single-copy orthologs among the protein sequences of Anolis carolinensis (GCA_000090745.2), Chelonia mydas (GCA_015237465.2), Danio rerio (GCA_000002035.4), Deinagkistrodon acutus [29], 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), and Alligator mississippiensis (GCA_000281125.4). The number of orthogroups of all species was 7,788.
Reuse potential
Venomous animals have fascinated and influenced humans since ancient times, and the venom gland is a special evolutionary mechanism that snakes have developed to adapt to their ecological environment [30]. In recent years, ecosystems have changed due to climate variations, and toxic species threaten not only humans but also native species and livestock [31, 32]. Therefore, it is crucial to collect genomic resources of venomous snakes and explore the formation mechanism of venom glands and venom production.
Genome assemblies of reptiles, including snakes, have always been challenging to generate. However, Xu et al. recently published an article on the origin of neurotoxins in the Elapidae family based on a high-quality genome assembly of the many-banded krait [29]. Using third-generation sequencing and Hi-C, Xu et al. assembled the many-banded krait genome to the chromosome level, achieving a BUSCO score of 94.6% and a scaffold N50 of 149.80 Mbp. Our assembly resulted in a BUSCO score of only 90.9%. Although our assembly did not achieve the same level of genome continuity as Xu et al., we obtained a relatively complete genome of the many-banded krait using stLRF second-generation sequencing data. Being sampled from a different geographic location provides a genomic resource for future research exploring the evolution and origin of reptilian species, including snakes.
Our data can be combined with already published and new venomous snake genome data to reconstruct the evolutionary history of venomous snakes and other reptiles. Our genome data can also be used in venomics research to explore toxic gland genes and the mechanism of toxic gland production.
Data Availability
The data that support the findings of this study have been deposited into CNGB Sequence Archive (CNSA) [33] of China National GeneBank DataBase (CNGBdb) [34] with accession number CNP0004003. The data are also hosted in NCBI with accession number PRJNA934116. Additional data is available in the GigaDB repository [35].
List of abbreviations
GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; LINEs: Long Interspersed Nuclear Elements; LTRs: Long Terminal Repeats; SINEs: Short Interspersed Nuclear Elements; TE, transposable element.
Declarations
Consent for publication
All experimental procedures were approved by the Institutional Animal Care and Use Committee of Northeast Forestry University (2023048).
Competing Interests
The authors declare no conflict of financial interests.
Authors’ contribution
SH, SY, and YX designed and initiated the project. ZD, DY, and YG collected the samples. BL, LC, and YM performed the DNA extraction and data analysis. BL, LC, ZD and TL wrote the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the Fundamental Research Funds for the Central Universities (No. 2572020DY02) and the Guangdong Provincial Key Laboratory of Genome Read and Write (grant No. 2017B030301011). This work was also supported by China National GeneBank (CNGB).
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Reviewer name and names of any other individual's who aided in reviewer Xu Jiang
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
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Are the data and metadata consistent with relevant minimum information or reporting standards? See GigaDB checklists for examples http://gigadb.org/site/guide Yes
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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
Is the validation suitable for this type of data? No
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 In this work, Boyang Liu et al used a stLFR method for the assembly of a Bungarus multicinctus genome. This work is technique sound for the innovation application fo stLFR which is cost low and sequencing fast. Meanwhile, the B.multicinctus is a very important species in snake as its highly lethal venom. Before further consideration, I think there are some issues should be answered. 1. Suprenova was used for this work, as I know, the supernova can export the phased info of the assembly. And I also noticed that authors used the redundans (v0.14a) for the removal of reduancy. So I am very intereset at how many redundancy was removed, and I didn't find this part of information in the work. 2. For Table 1. I noticed that the total length of contig and scaffold are same(both are 1621955402) , but the GC content of these two assemblies are highly different (39.6% vs 37.8%). May authors give some more explanation or discussion? 3. For Table 2 and Table 3, the statistics were not consistency, please choose one. 4. In the abstract, authors declaimed that "and also provides a molecular basis for the genes of the venom glands." However, in the Fig. 2, i don't find the toxicity venom information. As my knowledge, these families/pathway were enriched in B.multicinctus genome. I think the authors can check their annotation and RNA-seq data. 5. Fig 4 is not suitable. The species selection is a little sloppy. 6. There is a high quality genome reference of B.multicinctus released and deposited at http://www.gpgenome.com/species/148. However, this snake is not from guangxi. And I think the author can compare it with this work, the paper can be fetched from https://doi.org/10.1016/j.apsb.2022.11.015. How to use the database, please refer to https://doi.org/10.1007/s11427-021-1968-7.
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Reviewer name and names of any other individual's who aided in reviewer Nazila Godfrey
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 The writing needs to be improved significantly.
Are all data available and do they match the descriptions in the paper? No
Additional Comments There is only one Biosample submitted for this project while in methods is mentioned samples. It is not clear if the biosample is for DNA or RNA. Following sequencing data depends on this too.
Are the data and metadata consistent with relevant minimum information or reporting standards? See GigaDB checklists for examples http://gigadb.org/site/guide No
Additional Comments Fasta files required for genome assembly and BUSCO files are missing.
Is the data acquisition clear, complete and methodologically sound? No
Additional Comments The genome assemblies that are used need to be properly cited.
Is there sufficient detail in the methods and data-processing steps to allow reproduction? No
Additional Comments
Is there sufficient data validation and statistical analyses of data quality? No
Additional Comments
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? No
Additional Comments
Any Additional Overall Comments to the Author Introduction needs to be elaborated heavily. The importance of study needs to be clarified. Why only %6 of the bite cases is treated with antivenom? Is that because bites usually happen in remote areas, or we don’t have antivenom? It is confusing. Why genome sequencing is necessary for designing an antivenom? Main content section needs to be expanded and elaborated. It is not clear why at the end of this section they made such a statement: “This suggests that the significant expansion of repeating elements is an important manifestation of species differences”. Explain in the text.
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Comments on revised manuscript The revised manuscript should not be satisfied with. For Q3, if authors can not decide which result to choose, they shoudl use repeatmasker to integrate all annotation. For Q4 and Q6, answers are feckless. If the author want to provide a certified data for sharing, they should ensure the quality. Authors enhanced the importance of their data for venom analysis, they should prove their value themselves. And as some chromosome-level high quailty krait genomes have been released, they should compare them at least in the phylogenetic analysis.
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Comments to the Author The many-banded krait, Bungarus multicinctus is a highly venomous snake distributed across South and Eastern Asia. To help better understand the evolution of B. multicinctus, and also provides a molecular basis for the understanding venom production a 1.51Gb in size reference genome was sequenced and described here. This data can be combined with already published and new venomous snake genome data to construct the evolutionary history of venomous snakes and other reptiles. After submission another group published a genome of a different B. multicinctus individual, but after clarification on some parts of the genome assembly process as well as mentioning the other genome, it is useful to share this data to the community.
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