Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection
2.2. Nucleic Acid Extraction and cDNA Synthesis
2.3. Oxford Nanopore Technologies SMg and TSC
2.4. Illumina TSC
2.5. Data Analysis
2.6. Ethics Statement
2.7. Data Availability
3. Results
3.1. Hybridization Time and Sample Pooling on the MinION
3.2. TSC and Taxonomic Binning
3.3. Viral Genome Coverage and Sequencing Depth
3.4. Viral Detection in Human Samples
3.5. Viral Detection in Animal Samples
3.6. ONT Accuracy
3.7. Coverage Depth of Clinically Relevant Viruses
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample ID | Sample Type | qPCR Target | Ct Value | Symptoms | Sampling Date |
---|---|---|---|---|---|
H1 | Flocked swab nasopharynx | Enterovirus | 25 | Fever, dyspnea and coughing | 09/2018 |
H2 | Fecal | Enterovirus | 30 | Chronic diarrhea | 05/2016 |
H3 | Fecal | Norovirus Enterovirus | 17 29 | Fever, vomiting and abdominal pain | 11/2018 |
H4 | Fecal | Enterovirus | 21 | Fever, diarrhea and abdominal pain | 10/2016 |
A1 | Blood plasma | PRSSV 1 | - | None | 12/2017 |
A2 | Blood plasma | PRSSV 1 | 26 (pool) | None | 10/2018 |
A3 | Blood plasma | PRSSV 1 | 25 (pool) | Respiratory | 10/2017 |
A4 | Nasal swab | SIV 2 | 19 | Respiratory (closed enteral system) | 10/2018 |
Type | Detected Viruses | 20 min (Pool) % | 20 h (Pool) % | 20 h (Individual) 1 % |
---|---|---|---|---|
Human samples (n = 4) | Coxsackievirus A22 | 0.001 | 0.004 | 0.192 |
Enterovirus A71 | 0.00004 | 0.00011 | 0.00028 | |
Enterovirus D68 | 0.003 | 0.027 | 0.775 | |
Norovirus GII.4 | 1.42 | 4.77 | 10.56 | |
Animal samples (n = 4) | Astrovirus wild boar (n = 2) | 0.002 | 0.010 | 0.001 |
Bocavirus pig | 0.001 | 0.007 | 0.003 | |
Influenza A virus * | 55.31 | 51.15 | 9.39 | |
Mamastrovirus 2 | 0.0004 | 0.0017 | 0.0003 | |
Pasivirus A1 | 0.002 | 0.010 | 0.053 | |
PERV 2 (n = 4) | 10.63 | 11.83 | 11.34 | |
Porcine astrovirus 4 | 0.001 | 0.008 | 0.001 | |
Porcine bocavirus H18 | 0.009 | 0.029 | 0.016 | |
Porcine enterovirus B | 0.0002 | 0.0002 | 4.75 × 10−5 | |
Porcine kobuvirus | 0.49 | 1.35 | 0.28 | |
Porcine respirovirus 1 3 | 0.019 | 0.064 | 0.033 | |
Porcine sapelovirus 1 | 0.0001 | 0.0019 | 0.0003 | |
PRRSV 4 (n = 3) | 9.75 | 6.82 | 15.75 | |
Rotavirus C * | 0.00004 | 0.00005 | 0.00007 | |
Ungulate tetraparvovirus 3 | 10.02 | 0.44 | 5.24 | |
Total viral reads % | 87.66 | 76.53 | 53.64 |
ID | GenBank | Reference Length | Reference | Genome Coverage (%) | Average Sequencing Depth | ||||
---|---|---|---|---|---|---|---|---|---|
M | MV | NV | M | MV | NV | ||||
H1 | MH341731.1 | 7345 | Enterovirus D68 | 89 | 100 | 96 | 2 | 3342 | 2664 |
H2 | ─ | ─ | No virus detected | ─ | ─ | ─ | ─ | ─ | ─ |
H3 | MK073885.1 | 7555 | Norovirus GII.4 | 99 | 100 | 100 | 55 | 44,148 | 374,921 |
H3 | LR027546.1 | 7410 | Enterovirus A71 | 20 | 39 | 40 | <1 | 1 | 4 |
H4 | DQ995647.1 | 7401 | Coxsackievirus A22 | 35 | 100 | 85 | <1 | 898 | 152 |
Average (Human Samples) | 61 | 85 | 80 | 14 | 12097 | 94,435 | |||
A1 | HM159246 | 8774 | PERV 1 C | 83 | 71 | 64 | 28 | 7536 | 608 |
A1 | NC_038546 | 5114 | Porcine hokovirus HK7 | 18 | 41 | 74 | 1 | 977 | 6893 |
A1 | NC_035180 | 5533 | Ungulate tetraparvovirus 3 | 100 | 100 | 96 | 773 | 35518 | 380,898 |
A2 | NC_038537 | 4786 | Bocavirus pig/SX/China | 43 | 59 | 82 | 5 | 143 | 425 |
A2 | NC_038538 | 5267 | Porcine bocavirus P18 | 39 | 56 | 61 | 5 | 130 | 479 |
A2 | HM159246 | 8774 | PERV 1 C | 100 | 100 | 92 | 408 | 37,379 | 22,903 |
A2 | NC_025402 | 15396 | Porcine respirovirus 1 | 79 | 85 | 98 | 9 | 75 | 980 |
A2 | GU067771.1 | 15098 | PRRSV 2 (Amervac) | 89 | 100 | 100 | 21 | 21,836 | 11,949 |
A3 | NC_016896 | 6707 | Astrovirus wild boar | 10 | 17 | 32 | 60 | 1 | 144 |
A3 | NC_030653 | 10908 | Atypical porcine pestivirus 1 | 100 | 20 | 100 | 69 | <1 | 36 |
A3 | NC_018226 | 6916 | Pasivirus A1 | 91 | 78 | 63 | 38 | 243 | 478 |
A3 | HM159246 | 8774 | PERV 1 C | 33 | 81 | 49 | 2 | 1627 | 62 |
A3 | KT344816.1 | 15095 | PRRSV 2 (GER09-613) | 99 | 100 | 96 | 85 | 17,451 | 8459 |
A4 | NC_016896 | 6707 | Astrovirus wild boar | 98 | 81 | 76 | 20 | 12 | 128 |
A4 | NC_027711 | 6327 | Dromedary astrovirus | 83 | 44 | 38 | 5 | 5 | 23 |
A4 | KY250316-23 | 13200 | Influenza A virus * | 100 | 100 | 100 | 1512 | 24,292 | 197,905 |
A4 | NC_034974 | 6347 | Mamastrovirus 2 | 83 | 39 | 51 | 4 | 5 | 22 |
A4 | NC_023675 | 6639 | Porcine astrovirus 4 | 99 | 82 | 65 | 18 | 12 | 151 |
A4 | NC_023636 | 6500 | Porcine astrovirus 5 | 46 | 41 | 76 | 1 | 1 | 4 |
A4 | NC_016647 | 5076 | Porcine bocavirus 5 | 0 | 0 | 12 | 0 | 0 | 4 |
A4 | HQ540591 | 9182 | PERV 1 A | 73 | 96 | 58 | 1 | 7 | 6 |
A4 | KY214435 | 8043 | Porcine enterovirus b | 94 | 21 | 85 | 5 | 0.4 | 6 |
A4 | NC_016769 | 8210 | Porcine kobuvirus | 99 | 100 | 99 | 152 | 1457 | 4056 |
A4 | NC_003987 | 7491 | Porcine sapelovirus 1 | 98 | 59 | 66 | 3 | 2 | 26 |
A4 | GU067771.1 | 15098 | PRRSV (Amervac) 2 | 20 | 93 | 100 | <1 | 10 | 66 |
A4 | MN102366-75 | 18286 | Rotavirus A pig * | 4 | 2 | 10 | <1 | <1 | <1 |
A4 | NC_003985 | 7117 | Teschovirus A | 23 | 41 | 62 | <1 | 1 | 13 |
Average (animal samples) | 67 | 63 | 71 | 120 | 5508 | 23,582 |
Detected Virus | Percentage Identity (%) | Average Sequencing Depth (MV) |
---|---|---|
Norovirus GII.4 | 99.89 | 44,148 |
Influenza A virus * | 99.88 | 24,292 |
Enterovirus D68 | 99.83 | 3342 |
Ungulate tateraparvovirus 3 | 99.37 | 35,518 |
PRRSV 1 | 98.95 | 17,451 |
Porcine kobuvirus | 98.71 | 1457 |
Average | 99.44 | 21,035 |
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Schuele, L.; Cassidy, H.; Lizarazo, E.; Strutzberg-Minder, K.; Schuetze, S.; Loebert, S.; Lambrecht, C.; Harlizius, J.; Friedrich, A.W.; Peter, S.; et al. Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples. Viruses 2020, 12, 1358. https://doi.org/10.3390/v12121358
Schuele L, Cassidy H, Lizarazo E, Strutzberg-Minder K, Schuetze S, Loebert S, Lambrecht C, Harlizius J, Friedrich AW, Peter S, et al. Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples. Viruses. 2020; 12(12):1358. https://doi.org/10.3390/v12121358
Chicago/Turabian StyleSchuele, Leonard, Hayley Cassidy, Erley Lizarazo, Katrin Strutzberg-Minder, Sabine Schuetze, Sandra Loebert, Claudia Lambrecht, Juergen Harlizius, Alex W. Friedrich, Silke Peter, and et al. 2020. "Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples" Viruses 12, no. 12: 1358. https://doi.org/10.3390/v12121358