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Table 1 Results of routine RT-PCR assays and Nanopore sequencing

From: Evaluation on the use of Nanopore sequencing for direct characterization of coronaviruses from respiratory specimens, and a study on emerging missense mutations in partial RdRP gene of SARS-CoV-2

Specimen types Routine RT-PCR test results Nanopore sequencing results
HCoV Ct value Flow cell type Run time Consensus sequence length
(No. of mapped reads)
Consensus accuracy
(Sanger sequence length)
1* VC SARS-CoV-2 N: 29.65
orf1b: ND
Flongle 23 m 394 bp (268) 100% (394 bp)
2* VC SARS-CoV-2 N: 35
orf1b: ND
Flongle 32 m 394 bp (99) 100% (394 bp)
3* NPS-TS SARS-CoV-2 N: 29.8
orf1b: 30.42
Flongle 29 m 394 bp (248) 100% (297 bp)
4* SP SARS-CoV-2 N: 35
orf1b: 35
Sequencing was not performed due to absence of PCR target band
5# pOS SARS-CoV-2 N: 28.8
orf1b: 28.66
Flongle 22 m 394 bp (895) 100% (394 bp)
6# pOS SARS-CoV-2 N: 28.1
orf1b: 28.41
Flongle 16 m 394 bp (932) 100% (394 bp)
7# pOS SARS-CoV-2 N: 32.96
orf1b: 33.94
MinION 3 h 18 m 394 bp (36) 100%
8# pOS SARS-CoV-2 N: 33.5
orf1b: 33.5
Sequencing was not performed due to absence of PCR target band
9# pOS SARS-CoV-2 N: > 35
orf1b: > 35
Sequencing was not performed due to absence of PCR target band
10# pOS SARS-CoV-2 N: > 35
orf1b: > 35
Sequencing was not performed due to absence of PCR target band
11# pOS SARS-CoV-2 N: > 35
orf1b: > 35
MinION Not detected
12 pOS SARS-CoV-2 N: 14.5
orf1b: 15.48
MinION 17 m 394 bp (3420) 100% (394 bp)
13 pOS SARS-CoV-2 N: 15.6
orf1b: 16.67
MinION 31 m 394 bp (3036) 100% (394 bp)
14 pOS SARS-CoV-2 N: 19.2
orf1b: 19.92
MinION 31 m 394 bp (3449) 100% (394 bp)
15 pOS SARS-CoV-2 N: 19.52
orf1b: 21.2
MinION 22 m 394 bp (2176) 100% (394 bp)
16 pOS SARS-CoV-2 N: 21.1
orf1b: 21.76
MinION 23 m 394 bp (2617) 100% (394 bp)
17 pOS SARS-CoV-2 N: 22.1
orf1b: 23.27
MinION 17 m 394 bp (2484) 100% (394 bp)
18 pOS SARS-CoV-2 N: 22.6
orf1b: 22.77
MinION 7 m 394 bp (99) 100%
19 pOS SARS-CoV-2 N: 22.67
orf1b: 22.86
MinION 16 m 394 bp (3826) 100% (394 bp)
20 pOS SARS-CoV-2 N: 24.23
orf1b: 24.81
MinION 43 m 394 bp (58) 100%
21 pOS SARS-CoV-2 N: 24.5
orf1b: 25.19
MinION 31 m 394 bp (126) 100%
22 pOS SARS-CoV-2 N: 24.7
orf1b: 24.9
MinION 17 m 394 bp (2362) 100% (394 bp)
23 pOS SARS-CoV-2 N: 24.94
orf1b: 26.07
MinION 34 m 394 bp (54) 100%
24 pOS SARS-CoV-2 N: 27.76
orf1b: 28.9
MinION 1 h 394 bp (1)§ 97.21%‡§
25 pOS SARS-CoV-2 N: 30.47
orf1b: 30.6
MinION 33 m 394 bp (1846) 100% (326 bp)
26 pOS SARS-CoV-2 N: 31.26
orf1b: 31.61
MinION 1 h 394 bp (2)§ 99.24%‡§
27 pOS SARS-CoV-2 N: 31.68
orf1b: 32.23
MinION Not detected
28 pOS SARS-CoV-2 N: 35
orf1b: 35
MinION Not detected
29 NPS HKU1 N/A MinION Not detected
30 NPS HKU1 N/A MinION 52 m 394 bp (3804) 100% (394 bp)
31 NPS HKU1 N/A MinION 5 m 394 bp (3572) 100% (394 bp)
32 NPS HKU1 N/A MinION 6 m 394 bp (3877) 100% (394 bp)
33 NPA 229E N/A MinION 53 m 394 bp (4045) 100% (394 bp)
34 NPS 229E N/A MinION 51 m 394 bp (3908) 100% (394 bp)
35 NPS 229E N/A MinION 40 m 394 bp (4044) 100% (394 bp)
36 NPS 229E N/A MinION 1 h 27 m 394 bp (3882) 100% (394 bp)
37 NPS 229E N/A MinION 53 m 394 bp (4076) 100% (394 bp)
38 NPS 229E N/A MinION 46 m 394 bp (3971) 100% (394 bp)
39 NPS 229E N/A MinION 36 m 394 bp (4062) 100% (394 bp)
40 NPS OC43 N/A MinION 52 m 394 bp (3556) 100% (394 bp)
41 TS OC43 N/A Sequencing was not performed due to absence of PCR target band
42 NPS OC43 N/A MinION 56 m 394 bp (4126) 100% (394 bp)
43 TS OC43 N/A MinION 1 h 8 m 394 bp (3977) 100% (394 bp)
44 NS OC43 N/A MinION 2 m 394 bp (3845) 100% (394 bp)
45 NS OC43 N/A MinION 46 m 394 bp (4120) 100% (394 bp)
46 NPS OC43 N/A MinION 1 m 394 bp (85) 100% (394 bp)
47 NPS OC43 N/A MinION 1 h 26 m 394 bp (1543) 100% (364 bp)
48 NPA OC43 N/A MinION 1 h 14 m 394 bp (4066) 100% (394 bp)
49 NPS OC43 N/A MinION 44 m 394 bp (3998) 100% (394 bp)
50 NPS OC43 N/A MinION 43 m 394 bp (4096) 100% (394 bp)
51 NPS OC43 N/A MinION 40 m 394 bp (4015) 100% (394 bp)
52 NPS NL63 N/A MinION 54 m 394 bp (4029) 100% (394 bp)
53 NPS NL63 N/A MinION 35 m 394 bp (4019) 100% (394 bp)
54 NPS NL63 N/A Flongle 47 m 394 bp (851) 100% (298 bp)
55 NPS NL63 N/A MinION 37 m 394 bp (4030) 100% (394 bp)
56 NPS NL63 N/A Sequencing was not performed due to absence of PCR target band
57 NPS NL63 N/A MinION 1 h 11 m 394 bp (3898) 100% (394 bp)
58 SP Not detected MinION Not detected
59 SP Not detected MinION Not detected
60 NPS Not detected MinION Not detected
61 pOS Not detected MinION Not detected
  1. N and orf1b correspond to nucleocapsid gene and open reading frame 1b, respectively. The results of BioFire® FilmArray® Respiratory 2 Panel were qualitative and therefore Ct values were not available
  2. *Collected during the first wave of COVID-19 outbreak (January to early March, 2020, n = 4)
  3. #Collected during the second wave of COVID-19 outbreak (mid-March to May, 2020, n = 7)
  4. Collected during the third wave of COVID-19 outbreak (late June onwards, 2020, n = 17)
  5. Sanger sequence was not available due to high level of background noise. The Nanopore read/ consensus sequence was compared to SARS-CoV-2 reference genome (NC_045512.2)
  6. §The number of Nanopore reads was insufficient for generating accurate consensus sequence (< 30×)
  7. Ct threshold cycle, HCoV human coronavirus, N/A not available, ND not done, NPA nasopharyngeal aspirate, NPS nasopharyngeal swab, NS nasal swab, pOS posterior oropharyngeal saliva, RT-PCR reverse transcription polymerase chain reaction, SP sputum, TS throat swab, VC virus culture
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