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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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Virology Journal

ISSN: 1743-422X