De Novo Venom-Gland Transcriptomics of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia—Next-Generation Sequencing, Functional Annotation and Toxinological Correlation
Abstract
:1. Introduction
2. Results and Discussion
2.1. Sequencing and De Novo Transcriptome Assembly
2.2. Toxin Gene Expression Profile
2.3. Profiling of Toxin Transcripts
2.4. Sequence Analysis and Phylogenetics of Three-Finger Toxins
2.5. Clinical Relevance and Antigencity of Three-Finger Toxins
2.6. Phospholipases A2
3. Conclusions
4. Materials and Methods
4.1. Preparation of Snake Venom-Gland Tissue
4.2. RNA Extraction and Purification
4.3. Filtration of Raw Sequenced Reads
4.4. De Novo Transcriptome Assembly
4.5. Clustering and Functional Annotation of Transcripts
4.6. Quantifying Transcript Abundance
4.7. Categorization of Transcripts
4.8. Multiple Sequence Alignment
4.9. Phylogenetic Analysis
4.10. Scale-Based B-Cell Epitope Prediction
4.11. Supporting Data
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Parameter | Output Statistics |
---|---|
Total raw reads | 57,606,566 |
Total clean reads | 54,140,326 |
Total clean nucleotides (nt) | 4,872,629,340 |
Q20 percentage | 98.60% |
N percentage | <0.01% |
GC percentage | 44.63% |
Contigs created | 126,790 |
Total length (nt) | 51,459,117 |
Mean length (nt) | 406 |
N50 | 921 |
Unigenes/transcripts assembled | 82,209 |
Total length (nt) | 69,679,280 |
Mean length (nt) | 848 |
N50 | 2073 |
Unigene/transcripts assembled (FPKM > 1) | 70,564 |
Unidentified | 45,616 (17.97%) |
-Redundancy (FPKM abundance/number of transcripts) | 3.8 |
Non-toxin | 24,852 (33.86%) |
-Redundancy (FPKM abundance/number of transcripts) | 13.16 |
Toxin | 96 (48.18%) |
-Redundancy (FPKM abundance/number of transcripts) | 4847.54 |
Toxin Family/ID | UniProt Accession Code | Species | Expression Abundance (%) |
---|---|---|---|
Three-Finger Toxin (3FTx) | 77.26 | ||
S-3FTX | 56.48 | ||
Short neurotoxin SN160 | Q8UW27 | H. hardwickii | 56.43 |
Short neurotoxin homolog NTL4 | Q9YGI8 | B. multicinctus | <0.01 |
Short neurotoxin OH-35 | Q53B49 | O. hannah | <0.01 |
3FTx | C6JUP5 | M. corallinus | <0.01 |
putative three-finger toxin precursor | F5CPD1 | M. altirostris | <0.01 |
Short neurotoxin OH-26 | Q53B52 | O. hannah | <0.01 |
Cytotoxin homolog 5V | Q9W716 | Naja atra | 0.02 |
Cytotoxin homolog 5 | Q91137 | Naja atra | 0.01 |
Cytotoxin A5 | P62375 | Naja atra | 0.01 |
Cardiotoxin-like protein BMLCL | Q9PW19 | B. multicinctus | <0.01 |
L-3FTX | 20.78 | ||
Long neurotoxin 2 | A3FM53 | H. hardwickii | 20.74 |
Alpha-bungarotoxin isoform A31 | P60615 | B. multicinctus | 0.02 |
Kappa-bungarotoxin | P01398 | B. multicinctus | 0.01 |
Long neurotoxin homolog NTL2 | Q9YGH9 | B. multicinctus | 0.01 |
Long chain neurotoxin 6 | U3FYQ0 | M. fulvius | <0.01 |
Neurotoxin BM10-1-like | Q70WS8 | B. multicinctus | <0.01 |
Long chain neurotoxin 2 | U3FAC0 | M. fulvius | <0.01 |
NC-3FTX | <0.01 | ||
Weak toxin 1 | Q8AY51 | B. candidus | <0.01 |
Phospholipase A2 | 18.88 | ||
Basic phospholipase A2 73 | Q8UW30 | H. hardwickii | 18.84 |
Acidic phospholipase A2 | P00606 | B. multicinctus | 0.03 |
Phospholipase A2 MALT0035C | F5CPF1 | M. altirostris | 0.01 |
Phospholipase A2 GL16-1 | Q8JFB2 | L. semifasciata | <0.01 |
Basic phospholipase A2 beta-bungarotoxin A1 chain | P00617 | B. multicinctus | <0.01 |
Phospholipase A2 pkP2 | Q8JFG2 | L. semifasciata | <0.01 |
Cysteine-rich Secretory Protein | 3.34 | ||
Cysteine-rich venom protein 2 | Q8UW11 | H. hardwickii | 3.34 |
Cysteine-rich secretory protein Bc-CRPb | F2Q6G2 | B. candidus | 0.01 |
Phospholipase A2 Inhibitor | 0.13 | ||
phospholipase A2 inhibitor-like | A0A6J1W4V4 | N. scutatus | 0.13 |
C-type Lectin | 0.12 | ||
C-type lectin 1 | A3FM55 | H. hardwickii | 0.07 |
C-type lectin isoform 1 | H8PG89 | P. nigriceps | 0.04 |
Venom C-type lectin mannose binding isoform 4 | D2YVK4 | H. stephensii | 0.01 |
Kunitz-type Protease Inhibitor | 0.09 | ||
Putative Kunitz-type serine protease inhibitor | B2BS84 | A. labialis | 0.06 |
Kunitz-type protease inhibitor 1 | V8N7R6 | O. hannah | 0.01 |
Kunitz-type serine protease inhibitor homolog beta-bungarotoxin B1 chain | Q8AY46 | B. candidus | 0.01 |
Kunitz-type serine protease inhibitor PILP-2 | B4ESA3 | B. multicinctus | 0.01 |
Kunitz-type serine protease inhibitor spermatin | C1IC52 | W. aegyptia | <0.01 |
Kunitz-type serine protease inhibitor 28 | F8J2F3 | D. coronoides | <0.01 |
Protease inhibitor 4 | C1IC53 | W. aegyptia | <0.01 |
Kunitz-type serine protease inhibitor vestiginin-2 | A6MFL2 | D. vestigiata | <0.01 |
Kunitz-type serine protease inhibitor | P20229 | Naja naja | <0.01 |
Kunitz-type serine protease inhibitor 161 | F8J2F4 | D. coronoides | <0.01 |
Snake Venom Metalloproteinase | 0.08 | ||
Zinc metalloproteinase-disintegrin-like NaMP | A8QL59 | N. atra | 0.05 |
Porphyriacase-1 | B5KFV2 | P. porphyriacus | 0.01 |
Scutatease-1 | B5KFV7 | N. scutatus | 0.01 |
Zinc metalloproteinase-disintegrin-like BmMP | A8QL49 | B. fasciatus | <0.01 |
Zinc metalloproteinase-disintegrin-like MTP9 | F8RKV9 | D. coronoides | <0.01 |
Carinatease-1 | B5KFV1 | T. carinatus | <0.01 |
Snake venom metalloproteinase-disintegrin-like mocarhagin | Q10749 | N. mossambica | <0.01 |
Zinc metalloproteinase-disintegrin-like BfMP | A8QL48 | B. fasciatus | <0.01 |
Zinc metalloproteinase-disintegrin-like NaMP | A8QL59 | N. atra | <0.01 |
Stephensease-1 | B5KFV4 | H. stephensii | <0.01 |
Cystatin | 0.06 | ||
Cystatin | E3P6N8 | P. australis | 0.03 |
Cystatin | V8NX38 | O. hannah | 0.02 |
Cystatin-B | V8P5H9 | O. hannah | 0.01 |
Dipeptidyl Peptidase IV | 0.02 | ||
Venom dipeptidylpeptidase IV | A6MJI1 | T. carinatus | 0.02 |
Snake Venom Serine Protease | 0.01 | ||
Serine protease harobin | Q5MCS0 | H. hardwickii | 0.01 |
5’ Nucleotidase | <0.01 | ||
5’ nucleotidase | A6MFL8 | D. vestigiata | <0.01 |
5’-nucleotidase domain-containing protein 3 | V8P4R1 | O. hannah | <0.01 |
5’-nucleotidase | V8NYW9 | O. hannah | <0.01 |
Vascular Endothelial Growth Factor | <0.01 | ||
Vascular endothelial growth factor C | V8NCP7 | O. hannah | <0.01 |
Hyaluronidase | <0.01 | ||
Hyaluronidase | V8PHI0 | O. hannah | <0.01 |
Hyaluronidase | V8PFK9 | O. hannah | <0.01 |
Hyaluronidase | V8P1Z9 | O. hannah | <0.01 |
Phosphodiesterase | <0.01 | ||
2’,5’-phosphodiesterase 12 | V8PEM5 | O. hannah | <0.01 |
Waprin | <0.01 | ||
Supwaprin-a | B5KGY9 | A. superbus | <0.01 |
Natriuretic Peptide | <0.01 | ||
Natriuretic peptide Oh-NP | D9IX98 | O. hannah | <0.01 |
Natriuretic peptide Na-NP | D9IX97 | N. atra | <0.01 |
Cobra Venom Factor | <0.01 | ||
A.superbus venom factor 1 | Q0ZZJ6 | A. superbus | <0.01 |
Nerve Growth Factor | <0.01 | ||
NGF-Hop-5 | R4G2H9 | H. bungaroides | <0.01 |
Venom nerve growth factor 1 | Q3HXY6 | N. scutatus | <0.01 |
Aminopeptidase | <0.01 | ||
Aminopeptidase N | V8NGF6 | O. hannah | <0.01 |
Neprilysin | <0.01 | ||
Neprilysin | V8NQ76 | O. hannah | <0.01 |
L-amino-acid Oxidase | <0.01 | ||
L-amino-acid oxidase | A8QL51 | B. multicinctus | <0.01 |
Acetylcholinesterase | <0.01 | ||
Acetylcholinesterase | Q92035 | B. fasciatus | <0.01 |
Transcript ID | Toxin Gene Family/Annotated ID | UniProt Accession Code | Species | Transcript Length (aa) | Annotated ID Length (aa) | Coverage | Coverage to Mature Chain (%) |
---|---|---|---|---|---|---|---|
Three-Finger Toxin (3FTx) | |||||||
Lh_FTX01 | Short neurotoxin SN160 | Q8UW27 | H. hardwickii | 81 | 81 | 1–81 | 100 |
Lh_FTX02 | Short neurotoxin homolog NTL4 | Q9YGI8 | B. multicinctus | 71 | 86 | 16–86 | 100 |
Lh_FTX03 | Short neurotoxin OH-35 | Q53B49 | O. hannah | 63 | 86 | 15–85 | 100 |
Lh_FTX04 | 3FTx | C6JUP5 | M. corallinus | 62 | 79 | 15–78 | 98 |
Lh_FTX05 | putative three finger toxin precursor | F5CPD1 | M. altirostris | 66 | 82 | 21–82 | 100 |
Lh_FTX06 | Short neurotoxin OH-26 | Q53B52 | O. hannah | 62 | 78 | 15–77 | 98 |
Lh_FTX08 | Cytotoxin homolog 5V | Q9W716 | N. atra | 66 | 83 | 15–83 | 10 |
Lh_FTX10 | Cytotoxin A5 | P62375 | N. atra | 70 | 83 | 7–83 | 100 |
Lh_FTX11 | Cytotoxin A5 | P62375 | N. atra | 69 | 83 | 7–83 | 100 |
Lh_FTX12 | Cardiotoxin-like protein BMLCL | Q9PW19 | B. multicinctus | 97 | 103 | 7–103 | 100 |
Lh_FTX13 | Long neurotoxin 2 | A3FM53 | H. hardwickii | 93 | 93 | 1–93 | 100 |
Lh_FTX14 | Alpha-bungarotoxin isoform A31 | P60615 | B. multicinctus | 77 | 95 | 15–91 | 95 |
Lh_FTX15 | Kappa-bungarotoxin | P01398 | B. multicinctus | 72 | 87 | 15–86 | 94 |
Lh_FTX16 | Long neurotoxin homolog NTL2 | Q9YGH9 | B. multicinctus | 81 | 87 | 8–87 | 100 |
Lh_FTX17 | Long chain neurotoxin 6 | U3FYQ0 | M. fulvius | 72 | 84 | 14–84 | 100 |
Lh_FTX18 | Neurotoxin BM10-1-like | Q70WS8 | B. multicinctus | 66 | 84 | 15–84 | 100 |
Lh_FTX19 | Long chain neurotoxin 2 | U3FAC0 | M. fulvius | 99 | 87 | 4–84 | 96 |
Lh_FTX20 | Weak toxin 1 | Q8AY51 | B. candidus | 70 | 86 | 17–86 | 100 |
Phospholipase A2 | |||||||
Lh_PLA01 | Basic phospholipase A2 73 | Q8UW30 | H. hardwickii | 146 | 146 | 1–146 | 100 |
Lh_PLA02 | Acidic phospholipase A2 | P00606 | B. multicinctus | 132 | 145 | 14–145 | 100 |
Cysteine-rich Secretory Protein | |||||||
Lh_CRP01 | Cysteine-rich venom protein 2 | Q8UW11 | H. hardwickii | 238 | 238 | 1–238 | 100 |
C-type Lectin | |||||||
Lh_SCL01 | C-type lectin 1 | A3FM55 | H. hardwickii | 164 | 164 | 1–164 | 100 |
Lh_SCL02 | C-type lectin isoform 1 | H8PG89 | P. nigriceps | 172 | 157 | 1–157 | 100 |
Lh_SCL03 | Venom C-type lectin mannose binding isoform 4 | D2YVK4 | H. stephensii | 164 | 165 | 1–164 | 99 |
Kunitz-type Serine Protease Inhibitor | |||||||
Lh_KUN01 | Putative Kunitz-type serine protease inhibitor | B2BS84 | A. labialis | 249 | 252 | 1–252 | 100 |
Lh_KUN02 | Kunitz-type protease inhibitor 1 | V8N7R6 | O. hannah | 515 | 506 | 1–506 | 100 |
Lh_KUN03 | Kunitz-type serine protease inhibitor homolog beta-bungarotoxin B1 chain | Q8AY46 | B. candidus | 86 | 85 | 1–84 | 98 |
Lh_KUN04 | Kunitz-type serine protease inhibitor PILP-2 | B4ESA3 | B. multicinctus | 66 | 83 | 1–82 | 98 |
Lh_KUN05 | Kunitz-type serine protease inhibitor spermatin | C1IC52 | W. aegyptia | 79 | 81 | 1–79 | 98 |
Lh_KUN06 | Kunitz-type serine protease inhibitor 28 | F8J2F3 | D. coronoides | 66 | 83 | 18–83 | 100 |
Lh_KUN08 | Kunitz-type serine protease inhibitor vestiginin-2 | A6MFL2 | D. vestigiata | 71 | 83 | 16–81 | 97 |
Lh_KUN09 | Kunitz-type serine protease inhibitor | P20229 | N. naja | 53 | 57 | 5–57 | 93 |
Snake Venom Metalloproteinase | |||||||
Lh_SMP09 | Carinatease-1 | B5KFV1 | T. carinatus | 575 | 608 | 28–596 | 98 |
Lh_SMP10 | Scutatease-1 | B5KFV7 | N. scutatus | 586 | 608 | 28–608 | 100 |
Lh_SMP19 | Zinc metalloproteinase-disintegrin-like NaMP | A8QL59 | N. atra | 590 | 621 | 28–618 | 98 |
Cystatin | |||||||
Lh_CYS01 | Cystatin | E3P6N8 | P. australis | 141 | 141 | 1–141 | 100 |
Lh_CYS02 | Cystatin | V8NX38 | O. hannah | 164 | 171 | 8–171 | 96 |
Dipeptidyl Peptidase IV | |||||||
Lh_DPP01 | Venom dipeptidylpeptidase IV | A6MJI1 | T. carinatus | 753 | 753 | 1–753 | 100 |
Snake Venom Serine Protease | |||||||
Lh_SSP01 | Serine protease harobin | Q5MCS0 | H. hardwickii | 265 | 265 | 1–265 | 100 |
5’ Nucleotidase | |||||||
Lh_NUC01 | 5’ nucleotidase | A6MFL8 | D. vestigiata | 559 | 559 | 1–559 | 100 |
Vascular Endothelial Growth Factor | |||||||
Lh_VGF01 | Vascular endothelial growth factor C | V8NCP7 | O. hannah | 421 | 421 | 1–421 | 100 |
Hyaluronidase | |||||||
Lh_HYA01 | Hyaluronidase | V8PHI0 | O. hannah | 481 | 469 | 19–469 | 96 |
Waprin | |||||||
Lh_WAP01 | Supwaprin-a | B5KGY9 | A. superbus | 64 | 75 | 16–75 | 100 |
Cobra Venom Factor | |||||||
Lh_CVF01 | A.superbus venom factor 1 | Q0ZZJ6 | A. superbus | 1652 | 1652 | 1–1652 | 100 |
Neprilysin | |||||||
Lh_NEP01 | Neprilysin | V8NQ76 | O. hannah | 750 | 675 | 16–675 | 98 |
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Tan, C.H.; Tan, K.Y. De Novo Venom-Gland Transcriptomics of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia—Next-Generation Sequencing, Functional Annotation and Toxinological Correlation. Toxins 2021, 13, 127. https://doi.org/10.3390/toxins13020127
Tan CH, Tan KY. De Novo Venom-Gland Transcriptomics of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia—Next-Generation Sequencing, Functional Annotation and Toxinological Correlation. Toxins. 2021; 13(2):127. https://doi.org/10.3390/toxins13020127
Chicago/Turabian StyleTan, Choo Hock, and Kae Yi Tan. 2021. "De Novo Venom-Gland Transcriptomics of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia—Next-Generation Sequencing, Functional Annotation and Toxinological Correlation" Toxins 13, no. 2: 127. https://doi.org/10.3390/toxins13020127
APA StyleTan, C. H., & Tan, K. Y. (2021). De Novo Venom-Gland Transcriptomics of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia—Next-Generation Sequencing, Functional Annotation and Toxinological Correlation. Toxins, 13(2), 127. https://doi.org/10.3390/toxins13020127