Nanopore MinION Sequencing Generates a White Spot Syndrome Virus Genome from a Pooled Cloacal Swab Sample of Domestic Chickens in South Africa
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Approval
2.2. Sample Collection
2.3. Nucleic Acid Extraction
2.4. Metagenomic Sequencing
2.4.1. Round A—First-Strand Synthesis with Reverse Transcriptase (RT)
2.4.2. Second-Strand Synthesis with Sequenase Version 2.0 DNA Polymerase
2.4.3. Round B—Random PCR Amplification
2.4.4. Round C—PCR Enrichment and Clean Up
2.4.5. Nanopore MinION Library Preparation and Sequencing
2.4.6. Genome Assembly and Annotation
2.5. Phylogenetic Analysis
2.6. Principal Coordinates Analysis (PCoA)
3. Results
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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WSSV–SA (GenBank Accession: OR636681) | Frame | Percent Protein Identity: GenBank Accession (Country) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Protein | Start (bp) | End (bp) | NC_003225.3 (China) | AP027290.1 (Japan) | AF369029.2 (Thailand) | AF440570.1 (Taiwan) | MF768985.1 (Australia) | KU216744.2 (Mexico) | MN840357.1 (USA) | |
Immediate-early protein | 629 | 1303 | Forward | 100.0 | 99.6 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
dUTPase | 18,927 | 20,312 | Reverse | 100.0 | 100.0 | 100.0 | 100.0 | 98.7 | 100.0 | 100.0 |
Envelope protein VP53B | 20,510 | 23,416 | Forward | 100.0 | 100.0 | 99.9 | 100.0 | 100.0 | 100.0 | 100.0 |
Ribonucleotide reductase large subunit | 57,563 | 60,109 | Reverse | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Ribonucleotide reductase small subunit | 65,794 | 67,035 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 99.8 | 100.0 |
Envelope protein VP32 | 72,090 | 72,926 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Envelope protein VP187 | 78,128 | 82,948 | Reverse | 100.0 | 100.0 | 100.0 | 100.0 | 99.8 | 99.9 | 100.0 |
DNA-binding protein VP15 | 83,105 | 83,290 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 90.9 | 100.0 | 100.0 |
Envelope protein VP124 | 83,398 | 86,982 | Reverse | 100.0 | 99.9 | 100.0 | 99.9 | 99.2 | 99.9 | 100.0 |
Capsid protein VP76 | 87,052 | 89,076 | Forward | 100.0 | 100.0 | 99.9 | 99.9 | 98.0 | 100.0 | 100.0 |
E3 ligase | 89,095 | 91,629 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 99.9 | 99.8 | 100.0 |
Envelope protein VP41A | 98,576 | 99,454 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | NE* | 100.0 | 100.0 |
Envelope protein VP52A | 99,491 | 100,951 | Forward | 100.0 | 100.0 | 98.8 | 100.0 | NE* | 100.0 | 100.0 |
Envelope protein VP41B | 101,003 | 101,905 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | NE* | 100.0 | 100.0 |
Envelope protein VP52B | 110,480 | 111,634 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 99.7 | 100.0 |
Envelope protein VP38 | 111,695 | 112,624 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Capsid protein VP136 | 118,623 | 122,282 | Forward | 100.0 | 99.9 | 99.9 | 100.0 | 99.8 | 99.8 | 100.0 |
Capsid protein VP190 | 133,055 | 137,752 | Reverse | 100.0 | 99.9 | 99.9 | 98.7 | 99.4 | 99.8 | 100.0 |
Envelope protein VP14 | 137,706 | 137,999 | Reverse | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | NE* |
Putative TATA-box bind protein | 141,117 | 143,792 | Forward | 100.0 | 99.9 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Envelope protein VP39A | 143,779 | 145,038 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 91.3 | 100.0 | 100.0 |
Capsid protein VP51C | 145,063 | 146,463 | Forward | 100.0 | 100.0 | 99.8 | 100.0 | 100.0 | 100.0 | 100.0 |
Envelope protein VP26 | 147,361 | 147,975 | Reverse | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Envelope protein VP16 | 152,071 | 152,424 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 95.1 | 100.0 | 100.0 |
Envelope protein VP56 | 153,372 | 154,769 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 99.8 |
Envelope protein VP90 | 155,603 | 158,173 | Reverse | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Envelope protein VP11 | 160,758 | 162,059 | Reverse | 100.0 | 100.0 | 100.0 | 100.0 | NE* | 99.8 | 100.0 |
Envelope protein VP39 | 162,082 | 162,933 | Reverse | 100.0 | 100.0 | 100.0 | 100.0 | NE* | 100.0 | 100.0 |
Envelope protein VP31 | 162,937 | 163,722 | Reverse | 100.0 | 99.6 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Capsid protein VP664 | 177,046 | 195,279 | Forward | 100.0 | 100.0 | 99.9 | 99.9 | 99.8 | 100.0 | 100.0 |
Envelope protein VP12B | 195,419 | 195,625 | Reverse | 100.0 | 100.0 | 100.0 | 100.0 | 80.0 | 100.0 | 100.0 |
Anti-apoptosis protein | 198,046 | 199,011 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 99.7 | 100.0 | 100.0 |
Envelope protein VP19 | 208,379 | 208,744 | Reverse | 100.0 | 100.0 | 99.2 | 100.0 | 100.0 | 100.0 | 100.0 |
Capsid protein VP60B | 208,882 | 210,516 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 99.1 | 100.0 | 100.0 |
Envelope protein VP28 | 211,349 | 211,963 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 99.5 | 100.0 | 100.0 |
Putative protein kinase | 212,058 | 214,250 | Reverse | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Envelope and capsid protein VP95 | 222,182 | 224,584 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 99.9 | 99.8 | 100.0 |
Immediate-early protein | 240,901 | 244,632 | Reverse | 100.0 | 99.6 | 99.9 | 99.8 | 99.8 | 99.8 | 99.9 |
DNA polymerase | 265,017 | 272,072 | Forward | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Structural protein VP55 | 272,381 | 273,727 | Reverse | 100.0 | 100.0 | 100.0 | 99.8 | 99.8 | 99.8 | 100.0 |
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Share and Cite
Chauhan, R.P.; Fogel, R.; Limson, J. Nanopore MinION Sequencing Generates a White Spot Syndrome Virus Genome from a Pooled Cloacal Swab Sample of Domestic Chickens in South Africa. Microorganisms 2023, 11, 2802. https://doi.org/10.3390/microorganisms11112802
Chauhan RP, Fogel R, Limson J. Nanopore MinION Sequencing Generates a White Spot Syndrome Virus Genome from a Pooled Cloacal Swab Sample of Domestic Chickens in South Africa. Microorganisms. 2023; 11(11):2802. https://doi.org/10.3390/microorganisms11112802
Chicago/Turabian StyleChauhan, Ravendra P., Ronen Fogel, and Janice Limson. 2023. "Nanopore MinION Sequencing Generates a White Spot Syndrome Virus Genome from a Pooled Cloacal Swab Sample of Domestic Chickens in South Africa" Microorganisms 11, no. 11: 2802. https://doi.org/10.3390/microorganisms11112802