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Tres productos desde un diseño únicoque permite el
secuenciamiento
de genomas completosy el desarrollo de diagnósticos
moleculares
Mariana Leguía, Ph.D.
Directora, Laboratorio de Genómica
Pontificia Universidad Católica del Perú
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SARS-CoV-2: virus que causa COVID-19 (Corona Virus
Disease-2019)
Adapted
from:https://www.nytimes.com/interactive/2020/04/03/science/coronavirus
-genome-bad-news-wrapped-in-protein.html
https://www.nytimes.com/es/interactive/2020/04/30/science/coronavirus
-mutacion.html
https://www.nytimes.com/es/interactive/2020/04/30/science/coronavirus-mutacion.html
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Estrategias de trabajo para SARS-CoV-2
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Estrategias de trabajo para SARS-CoV-2
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Estrategias de trabajo para SARS-CoV-2
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Estrategias de trabajo para SARS-CoV-2
#
ORF 1ab RdRp Gene E Gene N Gene
China Instituto
PasteurOMS
Instituto
PasteurOMS OMS China CDC_N1 CDC_N2 CDC_N3
Ct Average Ct Average CT Average Ct Average Ct Average Ct
Average Ct Average Ct Average Ct Average Ct Average
125.40
25.0623.65
23.4924.56
24.5322.41
22.3322.22
22.5029.17
29.1522.65
22.6025.29
25.2624.30
23.5524.18
24.2824.72 23.32 24.50 22.25 22.79 29.13 22.55 25.23 22.79
24.38
240.46
40.7336.67
37.0438.08
38.8235.48
35.6331.54
31.6444.53
43.4336.20
35.5038.67
38.9936.11
37.2137.50
38.4141.00 37.41 39.57 35.79 31.73 42.34 34.80 39.31 38.30
39.32
328.96
28.9227.91
28.1728.07
27.9326.04
26.1526.04
26.0932.77
32.8226.60
26.5528.82
28.8128.52
28.5028.23
28.4428.88 28.43 27.79 26.26 26.13 32.88 26.50 28.80 28.48
28.66
421.56
21.4719.14
19.2321.72
21.5018.66
18.6519.14
19.4226.15
26.3519.37
19.2321.29
21.5321.45
21.3020.53
20.3421.37 19.32 21.29 18.65 19.70 26.55 19.09 21.77 21.14
20.15
531.13
33.0130.70
30.9530.76
31.5128.84
28.7630.06
30.0739.03
39.5131.11
31.1233.92
33.9733.04
33.3632.75
33.0534.89 31.20 32.26 28.69 30.09 40.00 31.13 34.01 33.68
33.35
636.68
37.4933.13
33.2435.02
35.0731.34
31.6731.33
31.3939.59
40.4032.88
32.6135.52
35.7935.95
36.0934.47
34.6638.30 33.35 35.13 32.00 31.46 41.22 32.34 36.07 36.23
34.84
Existing RT-qPCR protocols for SARS-CoV-2:
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Estrategias de trabajo para SARS-CoV-2
#
ORF 1ab RdRp Gene E Gene N Gene
China Instituto
PasteurOMS
Insituto
PasteurOMS OMS China CDC_N1 CDC_N2 CDC_N3
Ct Average Ct Average CT Average Ct Average Ct Average Ct
Average Ct Average Ct Average Ct Average Ct Average
125.40
25.0623.65
23.4924.56
24.5322.41
22.3322.22
22.5029.17
29.1522.65
22.6025.29
25.2624.30
23.5524.18
24.2824.72 23.32 24.50 22.25 22.79 29.13 22.55 25.23 22.79
24.38
240.46
40.7336.67
37.0438.08
38.8235.48
35.6331.54
31.6444.53
43.4336.20
35.5038.67
38.9936.11
37.2137.50
38.4141.00 37.41 39.57 35.79 31.73 42.34 34.80 39.31 38.30
39.32
328.96
28.9227.91
28.1728.07
27.9326.04
26.1526.04
26.0932.77
32.8226.60
26.5528.82
28.8128.52
28.5028.23
28.4428.88 28.43 27.79 26.26 26.13 32.88 26.50 28.80 28.48
28.66
421.56
21.4719.14
19.2321.72
21.5018.66
18.6519.14
19.4226.15
26.3519.37
19.2321.29
21.5321.45
21.3020.53
20.3421.37 19.32 21.29 18.65 19.70 26.55 19.09 21.77 21.14
20.15
531.13
33.0130.70
30.9530.76
31.5128.84
28.7630.06
30.0739.03
39.5131.11
31.1233.92
33.9733.04
33.3632.75
33.0534.89 31.20 32.26 28.69 30.09 40.00 31.13 34.01 33.68
33.35
636.68
37.4933.13
33.2435.02
35.0731.34
31.6731.33
31.3939.59
40.4032.88
32.6135.52
35.7935.95
36.0934.47
34.6638.30 33.35 35.13 32.00 31.46 41.22 32.34 36.07 36.23
34.84
Existing RT-qPCR protocols for SARS-CoV-2:
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Protocolos de secuenciaciónde genomas completos de
SARS-CoV-2
Alejandra García Glaessner
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Optimización de primers en singleplex
1Kb1Kb+ 1Kb 1 1532 4 145 6 9 127 108 11 13 16 301817 19 2920 21
24 2722 2523 26 281Kb+ 1Kb1Kb+ 1Kb 1Kb+
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Optimización de primers en multiplex
A B A B A B A B
Artic 30c 35c 40c
1Kb+ 1Kb
CT 33.0
CT 25.06
CT 37.5 CT 40.73
CT 28.92CT 21.47
A B A B A B A B1Kb+ 1Kb A B A B A B A B1Kb+ 1Kb
Artic 30c 35c 40c Artic 30c 35c 40c
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Rango Esperado: 1000-1287 bp
= Selección para verificación
9 10 11 12 13 14 15 16 1Kb1Kb+ 1Kb
0.5 kb
1.0 kb1.5 kb2.0 kb
24 25 26 27 28 29 301Kb+ 1Kb 1Kb 1Kb+
0.5 kb
1.0 kb1.5 kb2.0 kb
1 2 3 4 5 6 7 8 1Kb1Kb+ 1Kb
0.5 kb
1.0 kb1.5 kb2.0 kb
17 18 19 20 21 22 231Kb+ 1Kb 1Kb 1Kb+
0.5 kb
1.0 kb1.5 kb2.0 kb
Condiciones de amplificación son efectivas para un rango muy
amplio de muestras (Ct hasta 39)
Sample # CtSuggested
Cyc les
1 23.36 30
2 27.56 35
3 21.27 30
4 26.3 30
5 23.33 30
6 31.47 35
7 22.64 30
8 30.72 35
9 32.01 35
10 23.29 30
11 31.29 35
12 24.66 30
13 33.01 35
14 24.61 30
15 28.07 30
16 21.16 30
17 31.1 30
18 23.92 30
19 38.85 40
20 22.93 30
21 38.24 40
22 22.93 30
23 35.88 40
24 20.1 30
25 40.73 40
26 25.45 30
27 37.49 40
28 25.06 30
29 21.47 30
30 28.92 30
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Condiciones de amplificación son efectivas paragenerar genomas
completos de SARS-CoV-2
CT 21.16
CT 31.47
CT 38.88
AmpliconPool
16 17 18 19 20 21 22 23 24 25 26 27 28 29 301.5 kb
1.0 kb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1.5 kb
1.0 kb
1.5 kb
1.0 kb
30 PCRs Individuales – Amplificación de Genoma Completo de
SARS-COV-2
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Conclusiones
• Tenemos un protocolo de TilingPCR que amplifica
genomascompletos a partir de muestras con Cts de hasta 39
• Estamos completando el NGS y el análisis bioinformático
paraverificar resultados
• Beneficios: – método super sensible
– casi no hay contaminación de secuencias de hospedero
– por encima de 95% de reads útiles
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Equipo:Diana Juarez, Mariana Leguia, Alejandra Garcia
Glaessner
Preguntas?
[email protected]
