Development of a whole-genome sequencing method of human coronavirus OC43 in clinical samples based on amplicon sequencing technology

Abstract

Abstract:

Objective To develop an efficient sequencing method for directly obtaining the whole-genome sequence of human coronavirus OC43（HCoV-OC43） from clinical samples，providing a critical technical means for genome sequencing and phylogenetic analysis of HCoV-OC43. Methods Specific primers were designed for different subtypes of HCoV-OC43 by using amplicon-based sequencing technology，patch primers were further optimized to compensate for missing regions，enabling the amplification of two overlapping segments to reconstruct the full genome sequence，and quality control was performed on both primers and sequencing data. Additionally，the effects of different amplification reaction systems and viral loads on amplification efficiency and sequencing quality were compared. Results The established method efficiently retrieved whole-genome sequences of HCoV-OC43，achieving genome coverage ≥98% and an average sequencing depth of ≥1 500×，with high specificity and sensitivity. Among 41 HCoV-OC43-positive clinical samples，31 genome sequences were successfully obtained，including 22 whole-genome sequences，8 N gene sequences，and 1 S gene sequence. Phylogenetic analysis revealed discrepancies between classifications based on different sequence fragments. Conclusion The proposed sequencing method is suitable for obtaining whole-genome sequences from HCoV-OC43-positive clinical samples with Ct values <32，which provides technical support and scientific data for molecular surveillance and the prevention and control of respiratory viruses.

Key words: Human coronavirus OC43, Whole-genome sequencing, Genotype

CLC Number:

R446

Yuqing ZHOU, Yujia HUO, Lu ZHANG, Xuechun ZHANG, Xiao WANG, Bing ZHAO. Development of a whole-genome sequencing method of human coronavirus OC43 in clinical samples based on amplicon sequencing technology[J]. OCCUPATION AND HEALTH, 2025, 41(23): 3200-3208.

Figures/Tables 10

References 31

[1]	HAMRE D, PROCKNOW J J. A new virus isolated from the human respiratory tract[J]. Proc Soc Exp Biol Med, 1966, 121(1):190-193.
[2]	MATTHEWS R. Virus taxonomy:Classification and nomenclature of viruses:Ninth report of the international committee on taxonomy of viruses[J]. Intervirology, 1979, 12(3-5):129-296.
[3]	SHANKAR A. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike[J]. Sci Adv, 2019, 5(4):eaav4580.
[4]	LAU S K P, LEE P, TSANG A K L, et al. Molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination[J]. J Virol, 2011, 85(21):11325-11337.
[5]	ST-JEAN J R, JACOMY H, DESFORGES M, et al. Human respiratory coronavirus oc43:Genetic stability and neuroinvasion[J]. J Virol, 2004, 78(16):8824-8834.
[6]	OONG X Y, NG K T, TAKEBE Y, et al. Identification and evolutionary dynamics of two novel human coronavirus OC43 genotypes associated with acute respiratory infections:Phylogenetic,spatiotemporal and transmission network analyses[J]. Emerg Microbes Infect, 2017, 6(1):1-13.
[7]	YE R Z, GONG C, CUI X M, et al. Continuous evolution and emerging lineage of seasonal human coronaviruses:A multicenter surveillance study[J]. J Med Virol, 2023, 95(6):e28861.
[8]	COTTEN M, LAM T T, WATSON S J, et al. Full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus[J]. Emerg Infect Dis, 2013, 19(5):736-742.
[9]	VIJGEN L, KEYAERTS E, LEMEY P, et al. Circulation of genetically distinct contemporary human coronavirus OC43 strains[J]. Virology, 2005, 337(1):85-92.
[10]	TABOADA B T, ISA P, ESPINOZA M A, et al. Complete genome sequence of human coronavirus oc43 isolated from mexico[J]. Genome Announc, 2016, 4(6):e01256-16.
[11]	崔琪奇, 张雪纯, 袁洋, 等. 2012—2019年上海市浦东新区常见呼吸道冠状病毒分子流行特征研究[J]. 职业与健康, 2021, 37(8):1044-1047,1051.
[12]	QUICK J, GRUBAUGH N D, PULLAN S T, et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples[J]. Nat Protoc, 2017, 12(6):1261-1276.
[13]	SINGH V K, MANGALAM A K, DWIVEDI S, et al. Primer premier:Program for design of degenerate primers from a protein sequence[J]. Bio Techniques, 1998, 24(2):318-319.
[14]	RYCHLIK W. OLIGO 7 primer analysis software[J]. Methods Mol Biol Clifton NJ, 2007, 402:35-60.
[15]	TAMURA K, STECHER G, KUMAR S. Mega11:Molecular evolutionary genetics analysis version 11[J]. Mol Biol Evol, 2021, 38(7):3022-3027.
[16]	LEMOINE F, GASCUEL O. The bayesian phylogenetic bootstrap and its application to short trees and branches[J]. Molecular Biology and Evolution, 2024, 41(11):msae238.
[17]	FRIEDMAN N, ALTER H, HINDIYEH M, et al. Human coronavirus infections in israel:Epidemiology,clinical symptoms and summer seasonality of HCoV-HKU1[J]. Viruses, 2018, 10(10):515.
[18]	KOVACS D, MAMBULE I, READ J M, et al. Epidemiology of human seasonal coronaviruses among people with mild and severe acute respiratory illness in Blantyre,Malawi,2011-2017[J]. J Infect Dis, 2024: jiad587.
[19]	HAN S, XU B, FENG Q, et al. Multicenter analysis of epidemiological and clinical features of pediatric acute lower respiratory tract infections associated with common human coronaviruses in China,2014-2019[J]. Virol J, 2023, 20(1):229.
[20]	ZHANG Z, LIU W, ZHANG S, et al. Two novel human coronavirus OC43 genotypes circulating in hospitalized children with pneumonia in China[J]. Emerg Microbes Infect, 2022, 11(1):168-171.
[21]	PALERMO L M, UPPAL M, SKRABANEK L, et al. Features of circulating parainfluenza virus required for growth in human airway[J]. mBio, 2016, 7(2):e00235.
[22]	CARRILLO C, LU Z, BORCA M V, et al. Genetic and phenotypic variation of foot-and-mouth disease virus during serial passages in a natural host[J]. J Virol, 2007, 81(20):11341-11351.
[23]	ATKINSON B, GRAHAM V, MILES R W, et al. Complete genome sequence of zika virus isolated from semen[J]. Genome Announc, 2016, 4(5):e01116-16.
[24]	HERRMANN E C, HABLE K A. Experiences in laboratory diagnosis of parainfluenza viruses in routine medical practice[J]. Mayo Clin Proc, 1970, 45(3):177-188.
[25]	HOULDCROFT C J, BEALE M A, BREUER J. Clinical and biological insights from viral genome sequencing[J]. Nat Rev Microbiol, 2017, 15(3):183-192.
[26]	DINWIDDIE D L, HARDIN O, DENSON J L, et al. Complete genome sequences of four novel human coronavirus oc43 isolates associated with severe acute respiratory infection[J]. Genome Announc, 2018, 6(21):e00452-18.
[27]	MAURIER F, BEURY D, FLÉCHON L, et al. A complete protocol for whole-genome sequencing of virus from clinical samples:Application to coronavirus OC43[J]. Virology, 2019, 531:141-148.
[28]	贾慧茹, 周婷, 高夫超. 一步法RT-PCR和两步法RT-PCR对蜜蜂病毒诊断研究的比较[J]. .应用昆虫学报, 2015, 52(2):351-355.
[29]	李锦潮. 两步法和一步法SHERLOCK检测方法的优化及其在甲型和乙型流感病毒检测中的应用[D]. 南京: 南方医科大学, 2024.
[30]	VIJGEN L, KEYAERTS E, MOËS E, et al. Complete genomic sequence of human coronavirus oc43:Molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event[J]. J Virol, 2005, 79(3):1595-1604.
[31]	张越. 人冠状病毒OC43分子流行病学与种系进化动力学特征研究[D]. 北京: 北京协和医学院, 2014.

序号	Pool 1		Pool 2
序号	名称	序列	名称	序列
1	400_1_F	CCCGCTTCACTGATCTCTTGTT	2_F	CGTGAAATTGTTATGAATGCAAGTCC
2	400_1_R	AGACTGCACACAACACTCTTGT	2_R	AAACATGTCCAAAACCGCCAC
3	400_3_F	GCATGTGGCCTATCAGTTATATATGATTG	4_F	CATTATTTCCTACTTGGAGTCAGGAACT
4	400_3_R	TGCCTTCATTTCTTGCAATGTCTT	4_R	TGGTGCAAGCAAAACCATCTATC
5	5_F	GCAGTTTGGTTACATTGATTGCGA	6_F	GTGTATTGGAGTCCAGCACGTAA
6	5_R	AGGTACAGAGCTTTGCAAATGAGA	6_R	TCTGCATAATCACAAAATGCTTGACC
7	11_F	TTTGATGAGGCTGGCGAAGAAG	8_F	TGCATGGTGCTTATATAGTAGTGGAG
8	11_R	GCATCACTGCCAAAACTGATCG	8_R	CTGCAACTTTAGGTGGTTCAGAAT
9	13_F	CTGGAAATGCATTAAATGTGATTTAGCTC	10_F	GCATGGAGAGTTCCTTGTGCT
10	13_R	TCACCTTTAACAAAACACACATTAGGT	10_R	TCTTCTTCAACATCACTTTCCTCAAGA
11	400_7_F	CTAGGTGTTTCAGATGTGTGGCA	12_F	GCCAGAATTTGTTAAAGTTTTGGGTC
12	400_7_R	GCAAGAGCAGTGCTAGTAGTAAAAG	12_R	TGGCACATCAACATCAATAAGTACCA
13	400_9_F	CTTTCTTGTTTTAAGATTGGACGTAGAAGA	400_8_F	TGGCTCAACATTTTAGTGATTTTGGAA
14	400_9_R	GGCTGTTCCTTAAATGTAACACGC	400_8_R	TGGCTTTCTGAACTTGACTAGGC
15	400_11_F	CCTTTTTACAGAAATTAGAGGATAATCCCC	400_14_F	GGTAAACAAATTGATGATCATCGTATCACT
16	400_11_R	GCTTTAGGCACATACAGACCCA	400_14_R	TTACCCTGTGTTCTCGCATCAG
17	15_F	TGTTATGTCTCTACAGGGGGCA	16_F	GGCAGCGCGTCTTTCCTTTA
18	15_R	AAATGTCGAAACAAATGCAACATCAT	16_R	AGTGAAATTAACACCATCAACAGTTAGC
19	17_F	CATAGATATATGCAGCCAGGATAAAGTTTT	400_18_F	GGCTGTAAGAAGTTCCTTTAATTTTGATC
20	17_R	AGCTTGCTTAAAAGTGAAATACGTACC	400_18_R	AATGCATAACAGCGTCCAGACC
21	400_19_F	TGATTTGACTGGGGCAATATGTGA	20_F	TCATGTTAAGTGTGAACAATCTTATCAGC
22	400_19_R	GGTTAACACCGATTTAAAAGTTTGTTTCA	20_R	CAACACCACATCACCTGTAGCT
23	21_F	TGACAGTCTTAATTCTAAGTTGGGTTTTG	400_21_F	ACTCAGCGTATTATTAAAGCCCAATTT
24	21_R	GACAAACTCTTAACGTATTTGGTTTCAC	400_21_R	TGATTTCTTTGGTTTCTTTGGCACC
25	23_F	TGTGGTTAAAGCTGTGCGAAAT	22_F	TAGTGGTGACAGCTCAGAGAGT
26	23_R	ACTGATTCTTAAAACCCACATCCTG	22_R	TCAGCGGATATTTTAATCCAGCCA
27	400_25_F	CAGGTGTGTTAAAGATTGTCATTGAATTG	24_F	TGTTGGTAAGATTGCACAGTGGA
28	400_25_R	TTTGAACAAAAGCCAGTGCCAC	24_R	ACCTCATAAACACATGTGCTGGT
29	400_27_F	GTGATGGACAGCGCACATATGA	26_F	TGCATTGATTGTGATTCTTATAAACCAGG
30	400_27_R	TCTGAATCAATAGAACAACTTTTACGAGC	26_R	TGTAACAGACGTACCAGTGTTAGC
31	400_29_F	GGTGTTCTGATTCAAAATTCTGCAAAG	400_28_F	TGGTACGTCTGTTACAGAAACTATGTT
32	400_29_R	ACAAACATCTTCAACGCTAACATCTC	400_28_R	AGCATCCACAGACCATATACAGGA
33	29_F	TCTTAAAGGGGGTGCAGTTTTTAGT	400_30_F	CAAATCAGATTTTCAGCTTCCCGTT
34	29_R	GTATAACACTGCACTCCATCAGCA	400_30_R	TGGACTACCATCGGCCATTGTA
35	31_F	TTCCAGAAGTGTTGCGAGAAGG	30_F	TGGCTCTACAGTGTTTAATGTCCC
36	31_R	GGGGTAAACTTGAAACACAAAAAGC	30_R	TACCCTCATCAGCTTCCTCACA
37	400_33_F	TGTGTTTCAAGTTTACCCCATACTTTC	400_32_F	TGTGAGGAAGCTGATGAGGGTA
38	400_33_R	CAAGCAGCCTCCCTATATGCAG	400_32_R	GTGCATTATCACACTTATCTCCGAAG
39	37_F	CAATCAAAACGCACTAGATTGTTTAAAGG	400_34_F	TTGAAGAAATGGCTCTCACTACTTTTATG
40	37_R	AGAAAACAAATCATGGTTAATGCTACGT	400_34_R	TGGATTAGTCATATCTGAAGCAGAACA
41	400_37_F	GTGGGCTCTGTCCAATGGATTT	34_F	GGTATTGTGAAAATGGTAAATCCTACTTCT
42	400_37_R	GCTTAACCAACAACATGGCCAAA	34_R	GAACCCTTAATGGTATAACTACTACGCA
43	39_F	CCTCAAATTAAGATAGTGCTTTTGTGCT	36_F	CATGGCTTTATGCTGCTATACTTAACAA
44	39_R	AAAGCAACACTCAGATCCGAAGT	36_R	GTTACATACATGAACATAGTCCATTTCACA
45	41_F	ATGAGGCGCGTTTTAGTGGTT	400_36_F	AACCACAAGGAGCCTTTCATGT
46	41_R	TCCTGGATAGTTTGAATCTGCCATAC	400_36_R	TCCAAAGACACACCAGTCATAGAAG
47	43_F	TGGGAAGATTGTTTATGCTATACTTAGTGA	400_39_F	GAATGGTCTTTGTTACATTACGTAGCA
48	43_R	GTGGTAGCATCGGGTTTAACAGT	400_39_R	CTTAGGAGGGCGCAATCCATTA
49	45_F	AGCACTGACACTACTGTTCAATCA	40_F	TCGTCTTGCTTAATTGCTTGCAAC
50	45_R	CGCAATGCATAGCAAAGATCCAA	40_R	CGGTCGCGTTCATAAGCAGAT
51	47_F	GGCTTAGTAGGCGTTTTAACACTTG	42_F	TTGTACCAGATAAAAGTGTTTATGACCAG
52	47_R	CAACAAAAGGCACACCATCCAC	42_R	GGATCTAACTCCAAAACAACAAAATTGC
53	49_F	CAAGCGGTGTAAAATTTCAAACAGTT	400_42_F	ATCTGCTTATGAACGCGACCG
54	49_R	AGTGTTGGCAGGACATTACGC	400_42_R	AACTAGTGGCCAGTTACAATCATCA
55	400_52_F	CGCCGCCTTATTAAAGATGTTGAC	400_45_F	CCTATTGCCAATTGTGTTAAAATGTTGT
56	400_52_R	CAACCTTATCACTTCTATACACATGTGAG	400_45_R	GCCCTTAATTGTACATCAGTAGATAAACC
57	400_54_F	GGACCTCATGAATTCTGTTCACAAC	46_F	GGCTGAACACGATTTCTTTACATTTG
58	400_54_R	GAGAAGAGCAGACCACGCAA	46_R	CACATCCTGGGGCTGCAATAA
59	400_56_F	AAGCCACAATATTCATTCAAGTTGGT	48_F	GGATGATCGGTGTATTATACATTGTGC
60	400_56_R	GCGCGATAATACACACCATTAGTC	48_R	GAGCTACCCTCTTTAAGCAGGC
61	57_F	GGTACAGGGAAGTCACATCTTGC	50_F	AAAGCCAGGCTCTATTATGAAGCA
62	57_R	GGTTCAAGTGTACCCTTGCTCA	50_R	CAACATGTCTCATGTTTTCGGGC
63	59_F	CATGAAAGTTCTAGTGCTGTAAATATGCA	52_F	AGATTGAAGATCTTAGTATACGTGCTCTT
64	59_R	AACCGCTATAACTCTTGCTACAATCT	52_R	AATCATCTACAAAACATCCAGCTCCT
65	400_62_F	CTTTGATGCTGAAGGTGCTCATG	54_F	AGTTCACTGATGAGTCCTTTTACAAGA
66	400_62_R	CGCCAACAACCATAGTAACCAGT	54_R	TCCACATCGGTCCATTTACAACTAG
67	63_F	CTCTTGTAGAGTCTTGCAGCGT	400_57_F	TGGAAAAGTTAAGCCACCACTTAAT
68	63_R	GGCTTCAAATGCTCAAAGGCTG	400_57_R	CTGGCCGCTGTGTATACAACA
69	65_F	TTGTGGAATACGTTCACCAAGCT	58_F	CTCGTATTCGTGCTAAGCATTATGTTT
70	65_R	AGCACCATTATCACGACCATCAA	58_R	CACGCTTAGCTGCAAAGTTCTG
71	67_F	CTGGGGAGTAATGGTAAACCCG	400_61_F	CTACATTGACCTTAGATAAAGTGCCAC
72	67_R	GACTTAACAAGAGTCACAAAATCATCCA	400_61_R	GGCTTCCACAACAAAATCAATTCCT
73	69_F	ACAGGCTGTATGATGAATGTTGCT	400_63_F	TGCAAAAGTAGGGCGTGAGATT
74	69_R	CAACACTGCCACCAAGAGCT	400_63_R	GGGTTGCCAATATCATAACACAAAGT
75	71_F	GTTTGGAACGGGGGTGCTTATA	64_F	TCTTCCTGGCTGTAATGGAGGT
76	71_R	AACTTCTTGAATAGCGACATCAACAC	64_R	GCCTGTCCTGTATAATGACCAGTC
77	73_F	CATAGCTCAACTCACGGATGCA	66_F	GCAGTAATACCTATGGTGTCTGCA
78	73_R	AATTTTGACAAGCCTTACGGAAATGA	66_R	AAGAGCTAATAACACGGCTTTGTG
79	75_F	GCTTAGGTTTTTATAACCCTCCTACCA	400_69_F	CTTATTTTATCACTGACGAGAAGAGTGG
80	75_R	CATTAACAAAAGTAAGGCTGCGATACA	400_69_R	ACTCGCATATTAACAGGTACAGCT
81	400_78_F	CCATTACCACTGGTTTTGATCTTAATTGT	70_F	AAGAACATAGGTGAGTACAATGTGAGT
82	400_78_R	TATCAGCAGCAGTGGGACATCT	70_R	GGGAATAAACCATATCATTAATCTGGTCTG
83	79_F	GCAGCAAGGTGTTTTTAGGTATGA	72_F	CAAACAGCGCCACACATTAGTC
84	79_R	ACGATCTAAAACATAATAAGTACCCAAACC	72_R	GCACCGAGCTTAACTATTTCAAGTC
85	81_F	AGTGAGTTCCCTGCTATAACTATAGGT	74_F	AGGCGATCTTCCCGAGAATGAT
86	81_R	CAGAGGCATGACATAATAGTGTGAAAG	74_R	GCCAGCTTTAGATGATATTTTACCAGAA
87	83_F	CACTGTTCAGCCAATTGCAGATG	76_F	CGTTCTGGTAGTAATGATATTTGGATGC
88	83_R	CTTATTCCAAGTAGAAGGATTAAACCTGC	76_R	TGACAATTAAGATCAAAACCAGTGGTAA
89	400_85_F	ATAGGCACTTGTCCTGCAGGTA	400_94_F	GCCGTAATCAAGGTAGATTTTGTGC
90	400_85_R	GCCAACTATTATAATAAGTCGCATTAACCT	400_94_R	ACATCAAGGACTGGTGGTTTTACA
91	87_F	GGTTTTAGAGACTACATAACAAACAGAACT	78_F	GCTTGTCAACCTCCGTACTGTT
92	87_R	GAAGGTATTTGAATTTCATACAAACCACC	78_R	AGCCGTTGGTAAGGAAATTAAAAGTATC
93	89_F	ACACAGTTGCAAGTAGCTAATAGTTT	80_F	GGTTTGGGTACTTATTATGTTTTAGATCGT
94	89_R	CCAAGCCCATTAATGCGATACTG	80_R	AGGATGACAAATCGTATGTGGGT
95	91_F	GCTCTTAATAACTTATTGCAACAACTCTCT	82_F	TGCATATTTTACAGACACTGGTTTTGT
96	91_R	AACCACTCTTAGGAGCTATACCTCT	82_R	TCCCAATTTAATGGAGAGGGCAC
97	95_F	GCTTGGTGCCGTAATCAAGGTA	400_84_F	TACCCAATGGCAGGAAGGTTG
98	95_R	ACATCATTGTAAAACTCATAAAACTGCCT	400_84_R	GGGGGCACTTATAAGTACCTGTAAA
99	99_F	GGTATTGGCTATTCTTTGGCAGATTT	88_F	GGTTATCGGTTTACTAATTTTGAGCCA
100	99_R	GGGTACAACATTCCCTCCTGAT	88_R	CTGCCTAGACAACCTAATACAGAGG
101	101_F	TGATGTCAATACCCCGGCTGA	90_F	GCTCTCAGAAAATCAGATCAGTGGA
102	101_R	CCTCTCTTACCAAAACACTGCTG	90_R	GAGCGGTGAGACGACCATTAAT
103	103_F	ACAGTTCCCCATTCTTGCAGA	92_F	GCTCCATATGGTTTGTATTTTATCCACTT
104	103_R	CTGAGGTGTCTTCAGTAAAGGGC	92_R	TTGCCTCCTGTAACCTATTCATTTCA
105			94_F	AGAAATGTGGTGGTTGTTGTGATG
106			94_R	GCGAATGCTGTGAAACCATTTATTAC
107			E-M-N_2_F	CAGTTTATATCTGGACTGCTGATGAAG
108			E-M-N_2_R	CCGTCAGGGTATGATAGTCCTCA
109			98_F	CCCAGAAACAAACAACTTGATGTGT
110			98_R	CGATTTCCAGAGGACGCTCTAC
111			100_F	CAGTTTAGAAATGTTCAAACCAGGGG
112			100_R	TACTTGGGTCCCGATCGACAAT
113			102_F	TAGTGCAGGATCGCGTAGTAGA
114			102_R	GGCTCGTCAGGATTCCCAGATA

序号	Pool 1		Pool 2
序号	名称	序列	名称	序列
1	400_1_F	CCCGCTTCACTGATCTCTTGTT	2_F	CGTGAAATTGTTATGAATGCAAGTCC
2	400_1_R	AGACTGCACACAACACTCTTGT	2_R	AAACATGTCCAAAACCGCCAC
3	400_3_F	GCATGTGGCCTATCAGTTATATATGATTG	4_F	CATTATTTCCTACTTGGAGTCAGGAACT
4	400_3_R	TGCCTTCATTTCTTGCAATGTCTT	4_R	TGGTGCAAGCAAAACCATCTATC
5	5_F	GCAGTTTGGTTACATTGATTGCGA	6_F	GTGTATTGGAGTCCAGCACGTAA
6	5_R	AGGTACAGAGCTTTGCAAATGAGA	6_R	TCTGCATAATCACAAAATGCTTGACC
7	11_F	TTTGATGAGGCTGGCGAAGAAG	8_F	TGCATGGTGCTTATATAGTAGTGGAG
8	11_R	GCATCACTGCCAAAACTGATCG	8_R	CTGCAACTTTAGGTGGTTCAGAAT
9	13_F	CTGGAAATGCATTAAATGTGATTTAGCTC	10_F	GCATGGAGAGTTCCTTGTGCT
10	13_R	TCACCTTTAACAAAACACACATTAGGT	10_R	TCTTCTTCAACATCACTTTCCTCAAGA
11	400_7_F	CTAGGTGTTTCAGATGTGTGGCA	12_F	GCCAGAATTTGTTAAAGTTTTGGGTC
12	400_7_R	GCAAGAGCAGTGCTAGTAGTAAAAG	12_R	TGGCACATCAACATCAATAAGTACCA
13	400_9_F	CTTTCTTGTTTTAAGATTGGACGTAGAAGA	400_8_F	TGGCTCAACATTTTAGTGATTTTGGAA
14	400_9_R	GGCTGTTCCTTAAATGTAACACGC	400_8_R	TGGCTTTCTGAACTTGACTAGGC
15	400_11_F	CCTTTTTACAGAAATTAGAGGATAATCCCC	400_14_F	GGTAAACAAATTGATGATCATCGTATCACT
16	400_11_R	GCTTTAGGCACATACAGACCCA	400_14_R	TTACCCTGTGTTCTCGCATCAG
17	15_F	TGTTATGTCTCTACAGGGGGCA	16_F	GGCAGCGCGTCTTTCCTTTA
18	15_R	AAATGTCGAAACAAATGCAACATCAT	16_R	AGTGAAATTAACACCATCAACAGTTAGC
19	17_F	CATAGATATATGCAGCCAGGATAAAGTTTT	400_18_F	GGCTGTAAGAAGTTCCTTTAATTTTGATC
20	17_R	AGCTTGCTTAAAAGTGAAATACGTACC	400_18_R	AATGCATAACAGCGTCCAGACC
21	400_19_F	TGATTTGACTGGGGCAATATGTGA	20_F	TCATGTTAAGTGTGAACAATCTTATCAGC
22	400_19_R	GGTTAACACCGATTTAAAAGTTTGTTTCA	20_R	CAACACCACATCACCTGTAGCT
23	21_F	TGACAGTCTTAATTCTAAGTTGGGTTTTG	400_21_F	ACTCAGCGTATTATTAAAGCCCAATTT
24	21_R	GACAAACTCTTAACGTATTTGGTTTCAC	400_21_R	TGATTTCTTTGGTTTCTTTGGCACC
25	23_F	TGTGGTTAAAGCTGTGCGAAAT	22_F	TAGTGGTGACAGCTCAGAGAGT
26	23_R	ACTGATTCTTAAAACCCACATCCTG	22_R	TCAGCGGATATTTTAATCCAGCCA
27	400_25_F	CAGGTGTGTTAAAGATTGTCATTGAATTG	24_F	TGTTGGTAAGATTGCACAGTGGA
28	400_25_R	TTTGAACAAAAGCCAGTGCCAC	24_R	ACCTCATAAACACATGTGCTGGT
29	400_27_F	GTGATGGACAGCGCACATATGA	26_F	TGCATTGATTGTGATTCTTATAAACCAGG
30	400_27_R	TCTGAATCAATAGAACAACTTTTACGAGC	26_R	TGTAACAGACGTACCAGTGTTAGC
31	400_29_F	GGTGTTCTGATTCAAAATTCTGCAAAG	400_28_F	TGGTACGTCTGTTACAGAAACTATGTT
32	400_29_R	ACAAACATCTTCAACGCTAACATCTC	400_28_R	AGCATCCACAGACCATATACAGGA
33	29_F	TCTTAAAGGGGGTGCAGTTTTTAGT	400_30_F	CAAATCAGATTTTCAGCTTCCCGTT
34	29_R	GTATAACACTGCACTCCATCAGCA	400_30_R	TGGACTACCATCGGCCATTGTA
35	31_F	TTCCAGAAGTGTTGCGAGAAGG	30_F	TGGCTCTACAGTGTTTAATGTCCC
36	31_R	GGGGTAAACTTGAAACACAAAAAGC	30_R	TACCCTCATCAGCTTCCTCACA
37	400_33_F	TGTGTTTCAAGTTTACCCCATACTTTC	400_32_F	TGTGAGGAAGCTGATGAGGGTA
38	400_33_R	CAAGCAGCCTCCCTATATGCAG	400_32_R	GTGCATTATCACACTTATCTCCGAAG
39	37_F	CAATCAAAACGCACTAGATTGTTTAAAGG	400_34_F	TTGAAGAAATGGCTCTCACTACTTTTATG
40	37_R	AGAAAACAAATCATGGTTAATGCTACGT	400_34_R	TGGATTAGTCATATCTGAAGCAGAACA
41	400_37_F	GTGGGCTCTGTCCAATGGATTT	34_F	GGTATTGTGAAAATGGTAAATCCTACTTCT
42	400_37_R	GCTTAACCAACAACATGGCCAAA	34_R	GAACCCTTAATGGTATAACTACTACGCA
43	39_F	CCTCAAATTAAGATAGTGCTTTTGTGCT	36_F	CATGGCTTTATGCTGCTATACTTAACAA
44	39_R	AAAGCAACACTCAGATCCGAAGT	36_R	GTTACATACATGAACATAGTCCATTTCACA
45	41_F	ATGAGGCGCGTTTTAGTGGTT	400_36_F	AACCACAAGGAGCCTTTCATGT
46	41_R	TCCTGGATAGTTTGAATCTGCCATAC	400_36_R	TCCAAAGACACACCAGTCATAGAAG
47	43_F	TGGGAAGATTGTTTATGCTATACTTAGTGA	400_39_F	GAATGGTCTTTGTTACATTACGTAGCA
48	43_R	GTGGTAGCATCGGGTTTAACAGT	400_39_R	CTTAGGAGGGCGCAATCCATTA
49	45_F	AGCACTGACACTACTGTTCAATCA	40_F	TCGTCTTGCTTAATTGCTTGCAAC
50	45_R	CGCAATGCATAGCAAAGATCCAA	40_R	CGGTCGCGTTCATAAGCAGAT
51	47_F	GGCTTAGTAGGCGTTTTAACACTTG	42_F	TTGTACCAGATAAAAGTGTTTATGACCAG
52	47_R	CAACAAAAGGCACACCATCCAC	42_R	GGATCTAACTCCAAAACAACAAAATTGC
53	49_F	CAAGCGGTGTAAAATTTCAAACAGTT	400_42_F	ATCTGCTTATGAACGCGACCG
54	49_R	AGTGTTGGCAGGACATTACGC	400_42_R	AACTAGTGGCCAGTTACAATCATCA
55	400_52_F	CGCCGCCTTATTAAAGATGTTGAC	400_45_F	CCTATTGCCAATTGTGTTAAAATGTTGT
56	400_52_R	CAACCTTATCACTTCTATACACATGTGAG	400_45_R	GCCCTTAATTGTACATCAGTAGATAAACC
57	400_54_F	GGACCTCATGAATTCTGTTCACAAC	46_F	GGCTGAACACGATTTCTTTACATTTG
58	400_54_R	GAGAAGAGCAGACCACGCAA	46_R	CACATCCTGGGGCTGCAATAA
59	400_56_F	AAGCCACAATATTCATTCAAGTTGGT	48_F	GGATGATCGGTGTATTATACATTGTGC
60	400_56_R	GCGCGATAATACACACCATTAGTC	48_R	GAGCTACCCTCTTTAAGCAGGC
61	57_F	GGTACAGGGAAGTCACATCTTGC	50_F	AAAGCCAGGCTCTATTATGAAGCA
62	57_R	GGTTCAAGTGTACCCTTGCTCA	50_R	CAACATGTCTCATGTTTTCGGGC
63	59_F	CATGAAAGTTCTAGTGCTGTAAATATGCA	52_F	AGATTGAAGATCTTAGTATACGTGCTCTT
64	59_R	AACCGCTATAACTCTTGCTACAATCT	52_R	AATCATCTACAAAACATCCAGCTCCT
65	400_62_F	CTTTGATGCTGAAGGTGCTCATG	54_F	AGTTCACTGATGAGTCCTTTTACAAGA
66	400_62_R	CGCCAACAACCATAGTAACCAGT	54_R	TCCACATCGGTCCATTTACAACTAG
67	63_F	CTCTTGTAGAGTCTTGCAGCGT	400_57_F	TGGAAAAGTTAAGCCACCACTTAAT
68	63_R	GGCTTCAAATGCTCAAAGGCTG	400_57_R	CTGGCCGCTGTGTATACAACA
69	65_F	TTGTGGAATACGTTCACCAAGCT	58_F	CTCGTATTCGTGCTAAGCATTATGTTT
70	65_R	AGCACCATTATCACGACCATCAA	58_R	CACGCTTAGCTGCAAAGTTCTG
71	67_F	CTGGGGAGTAATGGTAAACCCG	400_61_F	CTACATTGACCTTAGATAAAGTGCCAC
72	67_R	GACTTAACAAGAGTCACAAAATCATCCA	400_61_R	GGCTTCCACAACAAAATCAATTCCT
73	69_F	ACAGGCTGTATGATGAATGTTGCT	400_63_F	TGCAAAAGTAGGGCGTGAGATT
74	69_R	CAACACTGCCACCAAGAGCT	400_63_R	GGGTTGCCAATATCATAACACAAAGT
75	71_F	GTTTGGAACGGGGGTGCTTATA	64_F	TCTTCCTGGCTGTAATGGAGGT
76	71_R	AACTTCTTGAATAGCGACATCAACAC	64_R	GCCTGTCCTGTATAATGACCAGTC
77	73_F	CATAGCTCAACTCACGGATGCA	66_F	GCAGTAATACCTATGGTGTCTGCA
78	73_R	AATTTTGACAAGCCTTACGGAAATGA	66_R	AAGAGCTAATAACACGGCTTTGTG
79	75_F	GCTTAGGTTTTTATAACCCTCCTACCA	400_69_F	CTTATTTTATCACTGACGAGAAGAGTGG
80	75_R	CATTAACAAAAGTAAGGCTGCGATACA	400_69_R	ACTCGCATATTAACAGGTACAGCT
81	400_78_F	CCATTACCACTGGTTTTGATCTTAATTGT	70_F	AAGAACATAGGTGAGTACAATGTGAGT
82	400_78_R	TATCAGCAGCAGTGGGACATCT	70_R	GGGAATAAACCATATCATTAATCTGGTCTG
83	79_F	GCAGCAAGGTGTTTTTAGGTATGA	72_F	CAAACAGCGCCACACATTAGTC
84	79_R	ACGATCTAAAACATAATAAGTACCCAAACC	72_R	GCACCGAGCTTAACTATTTCAAGTC
85	81_F	AGTGAGTTCCCTGCTATAACTATAGGT	74_F	AGGCGATCTTCCCGAGAATGAT
86	81_R	CAGAGGCATGACATAATAGTGTGAAAG	74_R	GCCAGCTTTAGATGATATTTTACCAGAA
87	83_F	CACTGTTCAGCCAATTGCAGATG	76_F	CGTTCTGGTAGTAATGATATTTGGATGC
88	83_R	CTTATTCCAAGTAGAAGGATTAAACCTGC	76_R	TGACAATTAAGATCAAAACCAGTGGTAA
89	400_85_F	ATAGGCACTTGTCCTGCAGGTA	400_94_F	GCCGTAATCAAGGTAGATTTTGTGC
90	400_85_R	GCCAACTATTATAATAAGTCGCATTAACCT	400_94_R	ACATCAAGGACTGGTGGTTTTACA
91	87_F	GGTTTTAGAGACTACATAACAAACAGAACT	78_F	GCTTGTCAACCTCCGTACTGTT
92	87_R	GAAGGTATTTGAATTTCATACAAACCACC	78_R	AGCCGTTGGTAAGGAAATTAAAAGTATC
93	89_F	ACACAGTTGCAAGTAGCTAATAGTTT	80_F	GGTTTGGGTACTTATTATGTTTTAGATCGT
94	89_R	CCAAGCCCATTAATGCGATACTG	80_R	AGGATGACAAATCGTATGTGGGT
95	91_F	GCTCTTAATAACTTATTGCAACAACTCTCT	82_F	TGCATATTTTACAGACACTGGTTTTGT
96	91_R	AACCACTCTTAGGAGCTATACCTCT	82_R	TCCCAATTTAATGGAGAGGGCAC
97	95_F	GCTTGGTGCCGTAATCAAGGTA	400_84_F	TACCCAATGGCAGGAAGGTTG
98	95_R	ACATCATTGTAAAACTCATAAAACTGCCT	400_84_R	GGGGGCACTTATAAGTACCTGTAAA
99	99_F	GGTATTGGCTATTCTTTGGCAGATTT	88_F	GGTTATCGGTTTACTAATTTTGAGCCA
100	99_R	GGGTACAACATTCCCTCCTGAT	88_R	CTGCCTAGACAACCTAATACAGAGG
101	101_F	TGATGTCAATACCCCGGCTGA	90_F	GCTCTCAGAAAATCAGATCAGTGGA
102	101_R	CCTCTCTTACCAAAACACTGCTG	90_R	GAGCGGTGAGACGACCATTAAT
103	103_F	ACAGTTCCCCATTCTTGCAGA	92_F	GCTCCATATGGTTTGTATTTTATCCACTT
104	103_R	CTGAGGTGTCTTCAGTAAAGGGC	92_R	TTGCCTCCTGTAACCTATTCATTTCA
105			94_F	AGAAATGTGGTGGTTGTTGTGATG
106			94_R	GCGAATGCTGTGAAACCATTTATTAC
107			E-M-N_2_F	CAGTTTATATCTGGACTGCTGATGAAG
108			E-M-N_2_R	CCGTCAGGGTATGATAGTCCTCA
109			98_F	CCCAGAAACAAACAACTTGATGTGT
110			98_R	CGATTTCCAGAGGACGCTCTAC
111			100_F	CAGTTTAGAAATGTTCAAACCAGGGG
112			100_R	TACTTGGGTCCCGATCGACAAT
113			102_F	TAGTGCAGGATCGCGTAGTAGA
114			102_R	GGCTCGTCAGGATTCCCAGATA

序号	Pool 1			Pool 2
序号	名称	引物序列	序列位置	名称	引物序列	序列位置
1	2-F	CTTGCTTAATGCTCCAGAG	5236-5692	1-F	GGCAGGTTGTTGTTAATGG	5167-5569
2	2-R	TGACCAACCTTATCACCTAT		1-R	TTACCGCAAGAACAGTCC
3	3-F	CCTACTGCTTCCGTCTCA	9915-10318	4-F	GCTTCAGATATGACTAATCCAG	10083-10520
4	3-R	CCTTGTGGTTTGCCGTTA		4-R	AACAACCTGAGCATCCTTAT
5	6-F	GTTGTTGGTTAAGCATAAGC	11024-11432	5-F	AAGGCACTGTTTGTTGGA	10879-11229
6	6-R	AGCAACCCACTTAGCAATA		5-R	AGACCATTCCTACAAGCAAT
7	8-F	CAGGTTGTGACGAGTATATC	22937-23352	7-F	TACACAGGCGAAGGTCAA	22621-22957
8	8-R	ACCAGCATCTCCATGATTA		7-R	CGATATACTCGTCACAACCT
9	9-F	ATGAGCAGCCTGATGTCT	24732-25147	84_F	ATGGTATGTGTTTTTCCAGCATAACT	24802-25196
10	9-R	CTACCTACACACGAACCATT		84_R	TTAGTACCTGCAGGACAAGTGC
11	93_F	TTCCTGATTTTAGGGAAGAGTTGGA	27330-27730	10-F	GCATAGGTGAGCACTGTT	25282-25707
12	93_R	TGCAATGAATCAAAGACGAACTTAGTC		10-R	CGGAAGAGTTAGCGTGAA
13	11-F	AACCACCAGTCCTTGATG	27443-27939	86_F	TGCCAACCACAAGCATTTTTGG	25347-25747
14	11-R	ACCGTCAGAGTATGATAGTC		86_R	CGTAGTTGCATTTAATATTCCGAAATAGC
15	13-F	GTGCCGTAATCAAGGTAGA	28331-28816	12-F	GAATAGGTTACAGGAGGCAAT	27929-28428
16	13-R	CTTCATCAGCAGTCCAGATA		12-R	GATTACGGCACCAAGCAT
17	97_F	TCCTAAAGGAATGGAATTTTTCTTTGGG	28437-28837	96_F	TGCAGACACTGTGTGGTATGTG	28131-28531
18	97_R	AAAGATGACCGCGTATTATTGTGAC		96_R	AACATAAACAAACATACTGCGACTTGT

序号	Pool 1			Pool 2
序号	名称	引物序列	序列位置	名称	引物序列	序列位置
1	2-F	CTTGCTTAATGCTCCAGAG	5236-5692	1-F	GGCAGGTTGTTGTTAATGG	5167-5569
2	2-R	TGACCAACCTTATCACCTAT		1-R	TTACCGCAAGAACAGTCC
3	3-F	CCTACTGCTTCCGTCTCA	9915-10318	4-F	GCTTCAGATATGACTAATCCAG	10083-10520
4	3-R	CCTTGTGGTTTGCCGTTA		4-R	AACAACCTGAGCATCCTTAT
5	6-F	GTTGTTGGTTAAGCATAAGC	11024-11432	5-F	AAGGCACTGTTTGTTGGA	10879-11229
6	6-R	AGCAACCCACTTAGCAATA		5-R	AGACCATTCCTACAAGCAAT
7	8-F	CAGGTTGTGACGAGTATATC	22937-23352	7-F	TACACAGGCGAAGGTCAA	22621-22957
8	8-R	ACCAGCATCTCCATGATTA		7-R	CGATATACTCGTCACAACCT
9	9-F	ATGAGCAGCCTGATGTCT	24732-25147	84_F	ATGGTATGTGTTTTTCCAGCATAACT	24802-25196
10	9-R	CTACCTACACACGAACCATT		84_R	TTAGTACCTGCAGGACAAGTGC
11	93_F	TTCCTGATTTTAGGGAAGAGTTGGA	27330-27730	10-F	GCATAGGTGAGCACTGTT	25282-25707
12	93_R	TGCAATGAATCAAAGACGAACTTAGTC		10-R	CGGAAGAGTTAGCGTGAA
13	11-F	AACCACCAGTCCTTGATG	27443-27939	86_F	TGCCAACCACAAGCATTTTTGG	25347-25747
14	11-R	ACCGTCAGAGTATGATAGTC		86_R	CGTAGTTGCATTTAATATTCCGAAATAGC
15	13-F	GTGCCGTAATCAAGGTAGA	28331-28816	12-F	GAATAGGTTACAGGAGGCAAT	27929-28428
16	13-R	CTTCATCAGCAGTCCAGATA		12-R	GATTACGGCACCAAGCAT
17	97_F	TCCTAAAGGAATGGAATTTTTCTTTGGG	28437-28837	96_F	TGCAGACACTGTGTGGTATGTG	28131-28531
18	97_R	AAAGATGACCGCGTATTATTGTGAC		96_R	AACATAAACAAACATACTGCGACTTGT

测试样编号	Ct值	不同反应体系扩增后产物浓度（ng/μL）
测试样编号	Ct值	TaKaRa One Step RNA PCR Kit-AMV一步法	Illumina COVIDSeq Assay两步法
2389	21.81	16.3	>60
2656	22.87	24.7	>60
2645	22.98	26.5	>60
ARI-60	23.99	11.8	>60
2668	24.01	26.1	>60
2353	24.08	21.8	>60
ARI-38	24.70	1.6	>60
2665	24.74	2.9	>60
2376	25.90	14.0	>60
1997	26.15	5.0	>60
1575	27.34	5.7	54.0
ARI-272	28.09	4.4	41.1
2374	29.95	21.3	30.7
ARI-166	30.76	13.3	29.6
21-4	30.84	3.5	25.3
ARI-11	32.09	11.2	30.0
21-2	32.83	3.1	22.7
15-176	33.22	7.7	22.9
17-575	33.75	2.3	25.2
14-4075	34.25	3.5	25.2