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

Wai Sing Chan; Chun Hang Au; Ho Yin Lam; Candy Ling Na Wang; Dona Ngar-Yin Ho; Yuk Man Lam; Daniel Ka Wing Chu; Leo Lit Man Poon; Tsun Leung Chan; Jonpaul Sze-Tsing Zee; Edmond Shiu Kwan Ma; Bone Siu Fai Tang

doi:10.1186/s12985-020-01454-3

Virology Journal

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
Specimen types		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

^¶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
^*Collected during the first wave of COVID-19 outbreak (January to early March, 2020, n = 4)
^#Collected during the second wave of COVID-19 outbreak (mid-March to May, 2020, n = 7)
^†Collected during the third wave of COVID-19 outbreak (late June onwards, 2020, n = 17)
^‡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)
^§The number of Nanopore reads was insufficient for generating accurate consensus sequence (< 30×)
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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ISSN: 1743-422X

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