Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Description
2.2. Routine Diagnostic Approach
2.3. RNA Extraction and Purification
2.4. Pre-Treatment (Chloroform/Beads/Centrifugation)
2.5. Real-Time RT PCR
2.6. Enterovirus Genotyping
2.7. Nanopore Sequencing
2.8. Illumina MiSeq. Primer Design for Coxsackievirus A6 cDNA Synthesis
2.9. cDNA Synthesis for Illumina MiSeq
2.10. Illumina MiSeq Sequencing
2.11. Bioinformatic Analysis
2.12. Data Availability
3. Results
3.1. Sample E590
3.2. Sample E372
3.3. Sample E026
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Sample E590 | Sample E372 | Sample E026 | ||||||
---|---|---|---|---|---|---|---|---|
Domain | Nanopore (reads) | Illumina MiSeq (contigs) | Nanopore (reads) | Illumina MiSeq (contigs) | Nanopore (reads) | Illumina MiSeq (contigs) | ||
DRS | Genotype specific primers | DRS | Random hexamers | Oligo-dT primer | DRS | Random hexamers | Oligo-dT primer | |
Eukaryota | 8850 | 1 | 10 | 19 | 28 | 1,349,407 | 227 | 56 |
Bacteria | 199 | 38 | 12,967 | 12,411 | 14,125 | 65,408 | 45,096 | 11,004 |
Archaea | 0 | 0 | 5 | 1 | 0 | 6 | 7 | 3 |
Viruses | 16 | 17 | 315 | 64 | 40 | 1579 | 398 | 63 |
Total | 9065 | 56 | 13,297 | 12,495 | 14,193 | 1,350,992 | 45,728 | 11,126 |
Sample E590 | Sample E372 | Sample E026 | ||||||
---|---|---|---|---|---|---|---|---|
Run ID | E590-DRS | E590-MiSeq | E372-DRS | E372-MiSeqR6 | E372-MiSeqOdT | E026-DRS | E026-MiSeqR6 | E026-MiSeqOdT |
Technology | DRS | MiSeq (custom primers) | DRS | MiSeq (random hexamers) | MiSeq (oligo dT) | DRS | MiSeq (random hexamers) | MiSeq (oligo dT) |
Coxsackievirus A6 | 16* | 8 | − | − | − | − | − | − |
Coxsackievirus A9 | − | − | − | − | − | 1 | − | − |
Coxsackievirus B1 | − | − | − | − | − | 4 | − | − |
Coxsackievirus B2 | − | − | 1 | − | − | − | − | − |
Coxsackievirus B3 | − | 1 | 1 | − | − | 5 | − | − |
Coxsackievirus B5 | − | − | 1 | − | − | 1 | − | − |
Echovirus 18 | − | 4 | − | − | − | − | − | − |
Echovirus 25 | − | − | − | − | − | 12 * | 1 * | 1 * |
Echovirus 30 | − | − | 124 | 28 | 1 * | − | 9 | 2 |
Echovirus 7 | − | − | 6 | − | − | − | − | − |
Echovirus E12 | − | − | − | − | − | 5 | − | − |
Echovirus E18 | − | − | − | − | − | 1 | − | − |
Echovirus E9 | − | − | − | − | − | 1 | − | − |
Enterovirus B106 | − | − | − | − | − | 2 | − | − |
Enterovirus B80 | − | − | − | − | − | 1 | − | − |
Enterovirus B81 | − | − | − | − | − | 5 | − | − |
Enterovirus B85 | − | − | − | − | − | 2 | − | − |
Enterovirus B86 | − | − | − | − | − | 1 | − | − |
Enterovirus B88 | − | − | − | − | − | 4 | − | − |
Rhinovirus A | − | − | − | 7 | 8 | − | − | − |
Bovine arvovirus 3 | − | − | − | − | 1 | − | − | − |
Cactus virus X | − | − | − | − | − | 21 | 12 * | 5 |
Caprine arthritis encephalitis virus | − | − | − | − | − | − | 1 | − |
Carrot cryptic virus | − | − | − | − | − | − | 4 | 1 |
Caudovirales | − | − | − | 3 | 2 | − | 22 | 5 |
Escherichia virus FI | − | − | − | − | − | − | 1 * | 2 |
Escherichia virus phiX174 | − | − | − | 1 * | 1 | − | 2 | 1 |
Gokushovirus WZ−2015a | − | − | − | − | − | − | − | 1 |
Human gut gokushovirus | − | − | − | − | − | − | − | 1 |
Melon necrotic spot virus | − | − | − | 1 | − | 8 | 1 * | 1 * |
Pitaya virus X | − | − | − | − | − | − | 4 | − |
Schlumbergera virus X | − | − | − | − | − | − | 12 | − |
Tomato mosaic virus | − | − | − | 2 | 5 | 1274 * | 248 | 1 * |
Tomato mottle mosaic virus | − | − | − | − | − | − | 5 | 1 |
Unclass. bacterial viruses | − | − | − | 18 | 21 | − | 61 | 37 |
Uncultured Microviridae | − | − | − | − | − | − | 1 | 1 |
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Grädel, C.; Terrazos Miani, M.A.; Baumann, C.; Barbani, M.T.; Neuenschwander, S.; Leib, S.L.; Suter-Riniker, F.; Ramette, A. Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing. Viruses 2020, 12, 841. https://doi.org/10.3390/v12080841
Grädel C, Terrazos Miani MA, Baumann C, Barbani MT, Neuenschwander S, Leib SL, Suter-Riniker F, Ramette A. Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing. Viruses. 2020; 12(8):841. https://doi.org/10.3390/v12080841
Chicago/Turabian StyleGrädel, Carole, Miguel A. Terrazos Miani, Christian Baumann, Maria Teresa Barbani, Stefan Neuenschwander, Stephen L. Leib, Franziska Suter-Riniker, and Alban Ramette. 2020. "Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing" Viruses 12, no. 8: 841. https://doi.org/10.3390/v12080841
APA StyleGrädel, C., Terrazos Miani, M. A., Baumann, C., Barbani, M. T., Neuenschwander, S., Leib, S. L., Suter-Riniker, F., & Ramette, A. (2020). Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing. Viruses, 12(8), 841. https://doi.org/10.3390/v12080841