Background
Methods
Specimens
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 |
RNA extraction
Reverse transcription and pan-coronavirus PCR
Primer sequences | ||
---|---|---|
1st PCR | ||
Forward | 5′-GGN TGG GAY TAY CCN AAR TGY GA-3′ | 760 bp amplicon |
Reverse | 5′-RHG GRT ANG CRT CWA TDG C-3′ | |
2nd PCR | ||
Forward | 5′-GGT TGG GAC TAT CCT AAG TGT GA-3′ | 440 bp amplicon |
Reverse | 5′-CCA TCA TCA GAT AGA ATC ATC AT-3′ |
Reverse transcription & First PCR | ||||
---|---|---|---|---|
Master mix constituents per reaction | PCR profile | |||
5× RT buffer | 5 µL | |||
dNTP (10 mM) | 1 µL | 45 °C | 30 m | |
5× Q solution | 5 µL | 95 °C | 15 m | |
Forward primer (200 µM) | 0.5 µL | 94 °C | 30 s | |
Reverse primer (200 µM) | 0.5 µL | 45 °C | 30 s | 50× |
One-step enzyme mix | 1 µL | 72 °C | 1 m | |
RNase-free water | 2 µL | 72 °C | 10 m | |
RNA | 10 µL | 15 °C | Hold | |
Total | 25 µL |
Second PCR | ||||
---|---|---|---|---|
Master mix constituents per reaction | PCR profile | |||
10x PCR buffer | 5 µL | |||
MgCl2 (25 mM) | 3 µL | 95 °C | 10 m | |
dNTP (10 mM) | 1 µL | 95 °C | 15 s | |
Forward primer (10 µM) | 1 µL | 45 °C | 30 s | 35× |
Reverse primer (10 µM) | 1 µL | 72 °C | 30 s | |
PCR-grade water | 33.5 µL | 72 °C | 5 m | |
AmpliTaq Gold™ | 0.5 µL | 15 °C | Hold | |
Purified 1st PCR amplicon | 5 µL | |||
Total | 50 µL |
Primer sequences | ||
---|---|---|
Forward 1 | 5′-ATG GGN TGG GAY TAY CC-3′ | ~ 440 bp amplicon |
Forward 2 | 5′-GGA YTA YCC NAA RTG YGA-3′ | |
Reverse | 5′-CCA TCA TCA SWN ARN ATS AT-3′ |
Reverse transcription | |||
---|---|---|---|
Master mix 1 constituents per reaction | Temperature profile | ||
Random hexamer | 1 µL | 65 °C On ice | 5 m 1 m |
dNTP (10 mM) | 1 µL | ||
RNA template | 8 µL | ||
Total | 10 µL |
Master mix 2 constituents per reaction | RT profile | ||
---|---|---|---|
10x RT buffer | 2 µL | ||
MgCl2 (25 mM) | 4 µL | 25 °C | 10 m |
DTT (0.1 M) | 2 µL | 50 °C | 50 m |
RNaseOUT | 1 µL | 85 °C | 5 m |
SuperScript III reverse transcriptase | 1 µL | 15 °C | Hold |
Master mix 1 | 10 µL | ||
Total | 20 µL |
PCR | ||||
---|---|---|---|---|
Master mix constituents per reaction | PCR profile | |||
10x PCR buffer | 5 µL | |||
MgCl2 (25 mM) | 8 µL | |||
dNTP (10 mM) | 1 µL | 95 °C | 9 m | |
Forward primer 1 (200 µM) | 1 µL | 95 °C (1 °C/s) | 1 m | |
Forward primer 2 (200 µM) | 1 µL | 48 °C (1 °C/s) | 1 m | 40× |
Reverse primer (200 µM) | 1 µL | 72 °C (1 °C/s) | 1 m | |
PCR-grade water | 11.5 µL | 72 °C | 5 m | |
AmpliTaq Gold™ | 1.5 µL | 15 °C | Hold | |
RT product | 20 µL | |||
Total | 50 µL |
Sanger sequencing
Nanopore sequencing
Bioinformatics
Results
Nanopore sequencing results
Consensus building without SARS-CoV-2 reference genome
Partial RdRP sequence analysis of SARS-CoV-2
Time | SNV | Africa | America | Asia/Middle East | Europe | Oceania | Subtotal | Total |
---|---|---|---|---|---|---|---|---|
Dec 2019 | Absent | 0 | 0 | 17 | 0 | 0 | 17 | 17 |
Present | 0 | 0 | 0 | 0 | 0 | 0 | ||
Jan 2020 | Absent | 0 | 18 | 318 | 24 | 9 | 369 | 384 |
Present | 0 | 0 | 15 | 0 | 0 | 15 (3.91%) | ||
Feb 2020 | Absent | 1 | 108 | 418 | 122 | 17 | 666 | 688 |
Present | 0 | 2 | 18 | 1 | 1 | 22 (3.20%) | ||
Mar 2020 | Absent | 72 | 319 | 660 | 10,816 | 1006 | 12,873 | 13,435 |
Present | 22 | 63 | 21 | 407 | 49 | 562 (4.18%) | ||
Apr 2020 | Absent | 27 | 1,803 | 455 | 4233 | 219 | 6,737 | 7082 |
Present | 30 | 90 | 25 | 194 | 6 | 345 (4.87%) | ||
May 2020 | Absent | 0 | 118 | 87 | 330 | 15 | 550 | 567 |
Present | 1 | 2 | 6 | 6 | 2 | 17 (3.00%) | ||
Subtotal | Absent | 100 | 2366 | 1955 | 15,525 | 1266 | 21,212 | 22,173 |
Present | 53 | 157 | 85 | 608 | 58 | 961 | ||
Total | 153 | 2523 | 2,040 | 16,133 | 1324 | Global | ||
% with SNV | 34.64 | 6.22 | 4.17 | 3.77 | 4.38 | 4.33 |
SNVs | Amino acid changes | No. of sequences | Remarks |
---|---|---|---|
15319C > T | P627S | 1 | §End of conserved polymerase motif A (17/17), containing the classic divalent-cation-binding residue D618, related to RdRP fidelity |
15327G > A | M629I | 2 | |
15327G > C | M629I | 1 | |
15327G > T | M629I | 13 | |
15328C > T | L630F | 2 | |
15338T > C | M633T | 1 | |
15346C > A | L636I | 2 | |
15346C > T | L636F | 4 | |
15349G > C | V637L | 1 | |
15352C > T | L638F | 1 | |
15356C > T | A639V | 2 | |
15358C > A | R640S | 1 | |
15359G > A | R640H | 1 | |
15364C > A | H642N | 1 | §Zn binding site (6/8) |
15368C > T | T643I | 4 | |
15371C > T | T644M | 4 | |
15380G > T | S647I | 47 | |
15384G > T | L648F | 1 | |
15392G > A | R651H | 1 | |
15406G > A | A656T | 17 | |
15406G > T | A656S | 1 | |
15407C > T | A656V | 1 | |
15412G > T | E658* | 1 | |
15418G > T | A660S | 3 | |
15438G > T | M666I | 34 | §End of nsp8 interaction site (52/52) |
15439G > A | V667I | 5 | |
15444G > T | M668I | 3 | |
15448G > A | G670S | 1 | |
15448G > T | G670C | 1 | |
15451G > A | G671S | 2 | |
15452G > T | G671V | 2 | |
15460T > C | Y674H | 2 | |
15463G > T | V675F | 2 | |
15472G > T | G678C | 1 | |
15488G > T | G683V | 1 | §Conserved polymerase motif B (4/30), related to RdRP fidelity |
15491A > G | D684G | 1 | §Conserved polymerase motif B (5/30), related to RdRP fidelity |
15535G > T | A699S | 3 | §Conserved polymerase motif B (20/30), related to RdRP fidelity |
15538G > A | V700I | 1 | §Conserved polymerase motif B (21/30), related to RdRP fidelity |
15539T > C | V700A | 1 | §Conserved polymerase motif B (21/30), related to RdRP fidelity |
15553A > G | N705D | 1 | §Conserved polymerase motif B (26/30), related to RdRP fidelity |
15571G > T | D711Y | 1 | |
15574G > A | G712S | 1 | |
15575G > T | G712V | 1 | |
15586G > T | A716S | 1 | |
15589G > T | D717Y | 1 | |
15593A > G | K718R | 2 | |
15594G > T | K718N | 1 | |
15596A > G | Y719C | 2 | |
15598G > T | V720F | 1 | |
15602G > A | R721H | 1 | |
15613C > T | H725Y | 2 | |
15619C > T | L727F | 1 | |
15627G > T | E729D | 1 | |
15636T > A | Y732* | 1 | |
15638G > A | R733K | 1 | |
15640A > T | N734Y | 1 | |
15641A > C | N734T | 1 | |
15647A > G | D736G | 4 | |
15652G > T | D738Y | 2 | |
15656C > T | T739I | 1 | |
15664G > A | V742M | 1 | |
15665T > C | V742A | 1 | |
15665T > G | V742G | 1 | |
15668A > G | N743S | 1 | |
15672G > T | E744D | 8 | |
15682T > A | Y748N | 1 | |
15683A > T | Y748F | 1 | |
15685T > A | L749M | 1 | |
15688C > T | R750C | 1 | |
15689G > A | R750H | 1 | |
15696T > G | H752Q | 1 |