Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation during a Pandemic
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection and Preparation
2.2. Real-Time Polymerase Chain Reaction
2.3. Tiling-Based Polymerase Chain Reaction
2.4. Library Preparation and Next-Generation Sequencing
2.4.1. TruSeq Nano DNA Library Preparation
2.4.2. NEBNext Ultra II DNA Library Preparation
2.4.3. Nextera DNA Flex Library Preparation
2.5. Data Analysis
3. Results
3.1. Sample Characteristics
3.2. SARS-CoV-2 Sequencing with Limited Reagents and Unavailability of Stock during a Pandemic in a Country in Lockdown
3.3. SARS-CoV-2 Genome Assembly
3.4. Association between Ct Value and Genome Coverage in an Outbreak
3.5. Comparison of Library Preparation Methods
3.6. Implementation of Nextera Flex and Improvement of Process and Success Rate of Sequencing
3.7. Phylogenetic and Lineage Analysis for Near Full-Length Genomes
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Coverage (% of SARS-CoV-2 Genome) | ||||
---|---|---|---|---|
Sequence | TruSeq DNA Nano | NEBnext Ultra II | Nextera Flex | Ct Value |
KRISP_0002 | 97.5 | 98.3 | 98.5 | 24.0 |
KRISP_0004 | 99.5 | 98.3 | 98.1 | 24.1 |
KRISP_0019 | 97.4 | 89.9 | 90.2 | NA |
KRISP_0021 | 63.5 | 99.1 | 82.7 | 14.1 |
KRISP_0024 | - | 95.2 | 94.6 | 21.5 |
KRISP_0026 | - | 99.8 | 99.9 | 17.9 |
KRISP_0028 | - | 96.1 | 98.1 | 21.4 |
KRISP_0031 | - | 86.2 | 84.1 | 24.3 |
KRISP_016 | 99.8 | - | 99.2 | NA |
KRISP_017 | 99.9 | - | 99.9 | NA |
KRISP_006 | 99.5 | - | 96.3 | 21.0 |
KRISP_007 | 99.9 | - | 99.6 | 25.5 |
KRISP_003 | 25.3 | 92.4 | - | Undetermined |
KRISP_010 | 93.4 | 92.3 | - | 25.6 |
KRISP_014 | 81.9 | 70.6 | - | NA |
KRISP_013 | 63.4 | 73.0 | - | NA |
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Pillay, S.; Giandhari, J.; Tegally, H.; Wilkinson, E.; Chimukangara, B.; Lessells, R.; Moosa, Y.; Mattison, S.; Gazy, I.; Fish, M.; et al. Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation during a Pandemic. Genes 2020, 11, 949. https://doi.org/10.3390/genes11080949
Pillay S, Giandhari J, Tegally H, Wilkinson E, Chimukangara B, Lessells R, Moosa Y, Mattison S, Gazy I, Fish M, et al. Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation during a Pandemic. Genes. 2020; 11(8):949. https://doi.org/10.3390/genes11080949
Chicago/Turabian StylePillay, Sureshnee, Jennifer Giandhari, Houriiyah Tegally, Eduan Wilkinson, Benjamin Chimukangara, Richard Lessells, Yunus Moosa, Stacey Mattison, Inbal Gazy, Maryam Fish, and et al. 2020. "Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation during a Pandemic" Genes 11, no. 8: 949. https://doi.org/10.3390/genes11080949