Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Sample Preparation
2.3. qPCR Confirmation and Quantification
2.4. GridION Library Preparation and Sequencing
2.5. Bioinformatic Analysis
3. Results
3.1. Comparison of IDV Detection by MiSeq and qPCR
3.2. Comparison of Nanopore Sequencing Results with Previously Determined MiSeq Data
3.3. Comparison of IDV Detection by qPCR, MiSeq and Nanopore Sequencing
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample * | Cq Value | Copy Number (Per Reaction) | Number (%) of IDV Reads | Largest IDV Contig (bp) | Total Input Reads | ||||
---|---|---|---|---|---|---|---|---|---|
Nanopore (WIMP) | Nanopore (In-House) | MiSeq | Nanopore (In-House) | MiSeq | Nanopore | MiSeq | |||
129 | 16.99 | 6.25 × 107 | 321,638 (14.69) | 606,932 (27.72) | 2182 (1.48) | 2030 | 951 | 2,188,805 | 147,341 |
114 | 17.53 | 1.31 × 107 | 8 (<0.01) | ND | ND | N/A | N/A | 335,559 | 136,961 |
50 | 20.71 | 1.25 × 106 | 1088 (5.74) | 1625 (8.57) | 162 (1.1) | 1161 | 498 | 18,966 | 14,719 |
69 | 21.69 | 4.48 × 105 | 944 (7.83) | 2287 (18.97) | 1812 (0.69) | 980 | 842 | 12,053 | 263,262 |
42 | 22.04 | 2.86 × 105 | 608 (0.34) | 584 (0.33) | 26 (0.07) | 986 | 499 | 179,559 | 37,560 |
6 | 23.33 | 2.16 × 105 | 281 (1.44) | 350 (1.8) | 183 (0.08) | 1034 | 522 | 19,512 | 228,875 |
10 | 24.09 | 1.31 × 105 | 1656 (1.17) | 1650 (1.11) | 87 (0.2) | 1359 | 485 | 148,381 | 44,433 |
199 | 26.01 | 4.64 × 104 | 4211 (10.99) | 10861 (28.34) | 3250 (1.49) | 2308 | 552 | 38,329 | 218,800 |
T50 | 26.76 | 2.93 × 104 | 3 (<0.01) | ND | ND | N/A | N/A | 43,193 | 1,256,918 |
T129 | 28.20 | 8.00 × 103 | 403 (0.23) | 502 (0.28) | 34 (<0.01) | 843 | 327 | 177,706 | 444,979 |
70 | 28.71 | 5.70 × 103 | 2 (0.02) | ND | ND | N/A | N/A | 12,211 | 160,704 |
T10 | 29.15 | 5.50 × 103 | 29 (0.54) | ND | 455 (0.03) | N/A | 913 | 5,413 | 1,415,256 |
13 | 29.22 | 4.15 × 103 | 116 (0.10) | 152 (0.13) | 48 (0.04) | 626 | 470 | 119,073 | 132,847 |
32 | 33.60 | 2.18 × 102 | ND | ND | ND | N/A | N/A | 275,387 | 32,312 |
170 | 35.62 | 8.25 × 101 | 1080 (2.03) | 1991 (3.75) | 8167 (0.74) | 903 | 1,584 | 53,131 | 1,100,167 |
135 | 36.51 | 4.88 × 101 | 10 (<0.01) | ND | ND | N/A | N/A | 142,737 | 154,220 |
260 | 39.46 | 6.88 | 9 (<0.01) | ND | ND | N/A | N/A | 164,607 | 959,935 |
T30 | ND | - | 3 (<0.01) | ND | 2 (<0.01) | N/A | 249 | 139,037 | 1,156,213 |
T52 | ND | - | 51 (0.1) | 52 (0.1) | 497 (0.05) | 1616 | 1,341 | 51,180 | 930,628 |
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Zhang, M.; Huang, Y.; Godson, D.L.; Fernando, C.; Alexander, T.W.; Hill, J.E. Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples. Viruses 2020, 12, 814. https://doi.org/10.3390/v12080814
Zhang M, Huang Y, Godson DL, Fernando C, Alexander TW, Hill JE. Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples. Viruses. 2020; 12(8):814. https://doi.org/10.3390/v12080814
Chicago/Turabian StyleZhang, Maodong, Yanyun Huang, Dale L. Godson, Champika Fernando, Trevor W. Alexander, and Janet E. Hill. 2020. "Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples" Viruses 12, no. 8: 814. https://doi.org/10.3390/v12080814
APA StyleZhang, M., Huang, Y., Godson, D. L., Fernando, C., Alexander, T. W., & Hill, J. E. (2020). Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples. Viruses, 12(8), 814. https://doi.org/10.3390/v12080814