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Aplicaciones de la Biología Molecular
en Diagnóstico Clínico
Jacobo Zuñiga Castillo PhD
~48% Molecular
Diagnostics
~10% Applied Testing
~19% Pharma
~23% Academic Research
SALES QIAGEN’s adjusted net sales grew 6% CER in FY 2017
$1.42 bn
EMPLOYEES QIAGEN has been repeatedly recognized as an employer of choice with leading HR programs.
4,800
EXPERIENCE QIAGEN was founded in 1984 by scientists from the Heinrich Heine University in Düsseldorf, Germany.
1984
LOCATIONS QIAGEN maintains subsidiaries in 20 countries and has more than 70 sales offices worldwide. 35
CUSTOMERS QIAGEN serves a truly global customer base across various markets in life science and healthcare.
500,000
PATENTS QIAGEN invests ~10% of its sales into R&D to continuously drive innovation and growth.
>2,200
PRODUCTS QIAGEN markets a broad range of consumables and instruments for various customer needs.
500
Facts and figures 2018
3
QIAGEN‘s Global Reach
QIAGEN subsidiary
Distributor
Venlo
Shanghai Germantown
Hilden
Over 35 subsidiaries in more than 20 countries
Distributor network in 70 countries
Dynamic growth in top 7 emerging markets
47%
20%
32%
4
We help customers gain insights to improve healthcare and advance science
• Complex Sample
•Sample Preparation •Pure Genes •Results
•Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, DNA, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Salts, Polymerases, Centrioles, Immunoglobulins, DNA, Cytokines, Angiotensins, Chemokines, Bradykines, Plasma membranes, Ribosomes, Actin, Vesicles, Complement components, Nuclei, Rough endoplasmatic reticulum, Nucleoli, Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Carrier proteins
Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, DNA, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Salts, Polymerases, Centrioles, Immunoglobulins, DNA, Cytokines, Angiotensins, Chemokines, Bradykines, Plasma membranes, Ribosomes, Actin, Vesicles, Complement components, Nuclei, Rough endoplasmatic reticulum, Nucleoli, Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Carrier proteins
DNA Information/
Insights
Virus detected
5
Multi-channel approach based on modern technologies
SAMPLE TO INSIGHT
Sample Tech
Assay Tech
Bioinfor- matics
Insights Samples
Our Core Our Customers Our Channels
Field Sales /Service
Inside Sales
Telemarketing
eCampaigns
QIA-Webshop
QIAcabinet, B2B digital
Academia
Pharma
HID/Forensic/ Food/Vet
Customer
Customer
Customer
… and with a very strong focus on Customer Intimacy
Diagnostics Customer
Clinical Sales Force
6
7
8
Cost of Sequencing is falling fast
9
QIAGEN: el primero en revolucionar la extracción de DNA…
…mediante el concepto de mini columnas de alta calidad!!!
1993 1953 …...la época oscura……
Estructura de DNA descrita Y publicada
Purificación de DNA con ultracentrifugación
QIAGEN introduce la mini columna
Extracción con fenol cloroformo
Cell cycle Angiogenesis
P53
MDM2
Cyclins
CDKs
VEGF
bFGF
ANGPT
PDGF
IL8
TLR
IFNγ
TNFβ Ind
ivid
ual
p
arti
cip
ants
Inflammation
Biological Markers Define Biological Processes
Path
way
Cell cycle Angiogenesis Inflammation
Complete Biological Story Built on Pathway / Network Analysis
• Assessing Changes (often in diseased/treated samples)
VEGF Expression Angiogenesis
Non-Cancer Cancer
0 10 YES
0 0 ?
If VEGF does not change in expression, traditionally you might interpret as no Angiogenesis.
But what if FLT3 (the VEGF receptor) or a downstream signaling partner is up-regulated…and makes the signaling pathway active?
Angiogenic signals will be activated in the nucleus.
\ A complete biological story is based on assessing all participants
Differential (∂) Gene Expression Analysis
10,000
89
1
# genes
Focused Pathway
Single Gene
Transcriptome
384
30,000+
5
Discovery
Focused Biological Discovery / Validation
Validation
12
Where Does The RT2 Profiler PCR Array fit into my Research?
•84 Pathway-Specific Genes of Interest
•5 Housekeeping Genes
•Genomic DNA Contamination Control
•Reverse Transcription Controls (RTC)
•Positive PCR Controls (PPC)
•Any Gene (up to 4)
Anatomy of “Modified” RT2 Profiler PCR Array
96 well version (1 sample/PCR Array) (384 well version available (4 samples/PCR Array)
Algunas definiciones….
Definicion de PCR
PCR = Reacción en cadena de la Polimerasa
Método in vitro rápido, simple, sensible y versatil para
amplificar selectivamente secuencias/regiones de DNA/RNA
a partir de una muestra compleja de ácidos nucleicos.
aguja
Definición PCR
Técnica de Biología Molecular que permite la producción
de grandes cantidades de una secuencia específica de
DNA a partir de un templado de DNA (mezcla compleja de
DNA) empleando una reacción enzimática sin la
participación de un organismo vivo (in vitro)
PCR: Fundamento
En 1983 Kary Mullis desarrolló el procedimiento conocido como PCR (reacción en cadena de la polimerasa)
Se desarrolla en tres etapas:
• Desnaturalización del templado (95°C)
• Alineamiento de Primers (50-60°C)
• Elongación de la cadena (72°C)
Ventajas de un PCR •Rapidez
•Alta sensibilidad
•Método robusto
•Alta Especificidad
•Alta Fidelidad
•Flexibilidad de la metodología
•Standarización de los métodos
•Facilidad de uso
Limitaciones de la PCR
•Se necesita conocer la secuencia a amplificar
•El tamaño de los fragmentos amplificados es
relativamente pequeño
•La PCR estándar no es una técnica adecuada para
análisis cuantitativos
¿De dónde obtenemos el DNA para trabajar?
Las muestras que utilizamos son muy variadas y PEQUEÑAS: sangre, biopsias, cultivo celular, tejidos varios etc.,
En nuestro genoma hay pocas copias de cada gen, entonces NO SON SUFICIENTES para realizar estudios posteriores.
El DNA es una muestra compleja, ¿cómo podemos aislar específicamente las secuencias que nos interesan?
PROBLEMA OBVIO: ¿Cómo obtener MUCHAS copias IDENTICAS de los genes o secuencias que nos interesan?
Diagrama de flujo para una PCR
Obtención de la muestra y
estabilización del DNA
Lisis de la muestra
y purificación del DNA
PCR Análisis
de la PCR
Factores que afectan una PCR
•Diseño de los primers
•Calidad del templado de DNA: presencia de contaminantes e
inhibidores.
•Buffer en donde se conserva el templado de DNA
•Concentración de dNTPs
•Concentración de primers
•Concentración de magnesio
•Concentración de templado
•Temperatura de alineamiento
Materiales utilizados para la PCR
•Reactivos – Polimerasas – Primers – dNTPs – Buffer – Templado de DNA
•Equipo: Termocicladores
– Estandar – Gradiente – In situ – Tiempo Real
Duplex DNA
94oC Desnaturalizacipón y alineamiento de primers
Primers forward y Reverse
60oC
72oC Extension
Ciclo
Desnaturalización -- alineamiento -- Extensión
El ciclo de la PCR
Gen - Específicos
Random hexamers
Degenerados
Tipos de Primers
¿Cómo definimos a un primer? Fragmentos cortos de DNA que son complementarios a la región blanco, es decir, aquella que se desea amplificar
Primers
Herramientas para el diseño de primers
•MethPrimer
•Oligo Analizer
•Oligo calc
•Webprimer
Polimerasa
Sus características determinan el tipo de amplificación:
Procesividad: La “distancia” que la enzima permanece unida al DNA, determinan el tamaño del fragmento
Termoestabilidad: La estabilidad a altas temperaturas, determinan tamaño y rendimiento
Fidelidad: (actividad PROOFREADING): Corrección de errores al sintetizar fragmentos de DNA, procedimientos como clonación
Tipo de extremos: Dos tipos de extremos blunt o romos y overhang o cohesivos, determinan posibilidad de clonación
Templado de DNA
Algunas recomendaciones generales son las siguientes:
•El DNA o RNA debe de encontrarse íntegro
•Se debe evitar la contaminación de las muestras de RNA con DNA
•Las muestras deberán ser purificadas cuidadosamente para eliminar los
inhibidores de la PCR (iones, solventes orgánicos etc)
•Preferentemente las muestras de DNA se almacenan en Tris 10 nM pH 7.5
Fuente • plásmido
• genómico
• viral
• cDNA Cantidad
• 0.1-10ng plasmido
• 0.1-1ug gDNA
Calidad
Sin inhibidores
Características del DNA molde
SDS >0.005% (W/V)
Fenol >0.2% (V/V)
Etanol >1%
Isopropanol >1%
Acetato de Sodio > 5 mM
Cloruro de Sodio > 25 mM
EDTA > 0.5 mM
Hemoglobina >1 mg/ml
Heparina > 0.15 I.U./ml
PCR Inhibidores
SDS Phenol EtOH NaAc NaCl EDTA
- - - - - -
Efecto de Inhibidores
Hay que optimizar
– [Mg2+]
– Temperatura de alineamiento
– Concentración de aditivos
– Número de ciclos
– Secuencia de los primers
– Concentración de los templados
– Tiempos de alineamiento y extensión
[Mg2+] mM Temperatura
[Mg2+] y Temperatura
Factores que influyen en la especificidad de la PCR
•Cantidad inicial de templado
•Diseño de las primers
•Cationes contenidos en los buffers de reacción
•Generación inicial de productos no especificos por la acción de la taq
polimerasa.
Composición exclusiva del buffer de reacción
Destabilization
Templado Primer
Stabilization Stabilization
Mayor
especificidad
+
NH4+ Buffers
Desestabiliza la unión no especifica de primers
Comparación de dos tipos de buffer en relación a la concentración de magnesio
PCR: 150 ng DNA genómico,.
NH4+ buffer
M M 1.5 1.5 4 4 mM Mg2+
750 bp
Sin buffer NH4+
NH4+ Buffers
Desestabiliza la uniòn no especifica de primers
Dímeros de primers en NTC muestran reacciones no especìficas
NH4+ Buffer Buffer sin NH4
+
Gene: Human BAX cDNA: 100 pg – 10 ng Primers: Hs_Bax_Primer Assay Cycler: Rotor-Gene 3000
Señal de dìmeros de primers en
NTC
No hay señal de dimeros en NTC
Señal de dimeros de primers en NTC
Tecnicas de PCR • RAPD
• AFLP
• PCR de colonias
• SSCP PCR (análisis conformacional de polimorfismos de cadena sencilla)
• PCR Hot Start
• PCR Inverso
• PCR In situ
• PCR para fragmentos largos
• RT – PCR
• PCR en Tiempo Real
• PCR de una célula
• PCR Multiplex
RT-PCR
Transcriptasa Reversa PCR
RT-PCR
Las principales aplicaciones de esta técnica son:
• Análisis de expresión:
– Cuáles genes están encendidos
– Estimado de cuánto están encendidos
• Construcción de bibliotecas de expresión (cDNA)
El templado inicial es RNA total o RNA mensajero
¿Entonces que necesitamos para amplificar RNA?
Una enzima que transforme el RNA en DNA para poder hacer una PCR:
La Transcriptasa Reversa (Transcripción reversa de la reacción en cadena de la polimerasa)
Cuáles son los reactivos necesarios para una RT-PCR?:
•Transcriptasa Reversa •Oligos •RNAsa •Reactivos para PCR
Aplicaciones en genómica y expresión
Genómica Expresión
• Identificación de genes • Cambios en la secuencia: • Polimorfismos • Mutaciones • Deleciones • Inserciones • Clonación • Identificación de
promotores
• Función de los genes • Mecanismos celulares • Regulación génica • Patrones de expresión
• Diseño y evaluación molecular de tratamientos
• Detección/diagnóstico de enfermedades
PCR en TIEMPO REAL
DNA RNA Proteína Genes y Promotores
Genotipificación
Clonación de Genes y promotores
Expresión
Manifestaciones Físicas o Fenotipo Actividad Función
Genotipificación: Secuencia de Genoma Identificar genes Encontrar diferencias, mutaciones
Puntos de regulación: Ensayos in vivo e in vitro como tansfecciones, CHIPs etc,
Interacciones DNA-Proteína (cromatina), RNAi, miRNA, epigenética
¿Cuáles genes se están transcribiendo
? ¿Qué tanto se están
transcribiendo?
¿Son activas/funcionales? ¿Qué tan activas? ¿Se unen a otras
proteínas?
Dogma Central de la Biología Molecular: Aplicaciones
Cuantificación
DNA DNA
GEN A GEN B
TRANSCRIPCIÓN = EXPRESIÓN
4 COPIAS
9 COPIAS
DNA DNA
GEN A GEN B
TRANSCRIPCIÓN = EXPRESIÓN
7 COPIAS
2 COPIAS
Células Epiteliales Normales: Perfil de Expresión
Células Epíteliales Cancerosas: Perfil de Expresión
RNA RNA
Perfiles de expresión
Gen de Referencia
– Se utiliza un gen de referencia para normalizar él ensayo:
• El gen de referencia es un gen cuya expresión debe ser constante en cualquier
condición analizada (tratamientos, enfermedades etc)
– La normalización con un gen de referencia permite:
• Corregir variaciones por la cantiodad de muestra (sangre, tejido, etc)
• Corregir las variaciones de RNA agregado en cada reacción
• Corregir variaciones debido al pipeteo
• Corregir la eficiencia de la RT
• Recobro de RNA en el proceso de extracción etc.
DNA DNA
GEN A GEN B
TRANSCRIPCIÓN = EXPRESIÓN
4 COPIAS
9 COPIAS
DNA
GEN HK
DNA DNA
GEN A GEN B
TRANSCRIPCIÓN = EXPRESIÓN
7 COPIAS
2 COPIAS
DNA
GEN HK
2 COPIAS 2 COPIAS
Perfiles de expresión: Normalización
Células Epiteliales Normales: Perfil de Expresión
Células Epíteliales Cancerosas: Perfil de Expresión
Perfiles de expresión: Análisis
DNA DNA
GEN A GEN B
TRANSCRIPCIÓN = EXPRESIÓN
4 COPIAS 9 COPIAS
DNA
GEN HK
DNA DNA
GEN A GEN B
TRANSCRIPCIÓN = EXPRESIÓN
7 COPIAS 2 COPIAS
DNA
GEN HK
2 COPIAS 2 COPIAS
Células Epiteliales Normales: Perfil de Expresión
Células Epíteliales Cancerosas: Perfil de Expresión
Normalización = GOI/GHK
VN = 2 VN = 4.5 VN = 3.5 VN = 1
Normalización = GOI/GHK
Gru
po
Co
ntr
ol
Gru
po
Mu
est
ra
Calibradores
Nivel de expresión = Muestra
Calibrador
GEN A
GEN B
1.75
0.29
=
=
Un poco de historia… ¿Cómo se hacía antes?
PCR Punto Final
DOR 2.6 =
Muestra Calibrador
Extracción RNA RNA RT - PCR Gel Agarosa
¿Cuáles son las limitaciones de la PCR en punto final?
Limitaciones del PCR punto final utilizando sistemas de detección Basados en análisis electroforético
Bromuro de etidio no es cuantitativo,
Pobre precisión
Baja sensibilidad
Baja Resolución
Bajo rango dinámico (2 logs)
Resultados subjetivos
Procesamiento Post PCR (gel agarosa)
5 10 15 20 30 40 50 0
¿Es posible cuantificar?
¿Es cuantitativa la PCR punto final?
Detección con Bromuro de Etidio
PCR Punto Final
Can
tid
ad d
e D
NA
Número de Ciclos
PCR Cuantitativo: PCR en tiempo real
La PCR en Tiempo Real permite monitorear los productos de PCR en el instante en el que se generan durante
cada ciclo.
Principio de la PCR en Tiempo Real
En la PCR en Tiempo Real la detección de los productos de PCR se realiza midiendo la fluorescencia que emiten:
Fluorescencia =
Absorbe luz de baja longitud de onda y emite luz de alta longitud de onda
Producto ds PCR
Producto ds PCR
Principio de la PCR en Tiempo Real
En cada ciclo se incrementa la intensidad de la fluorescencia
Producto ds PCR
...... ...... ...... ......
1
2
4
8
Detección y monitoreo en Tiempo Real
PCR convencional (punto final)
Detección con Bromuro de
Etidio
NTC
Fase Plateau
Fase Log-linear
baseline region
Curva de amplificación de un PCR en Tiempor Real
Fase Exponential
PCR Punto Final
Bajo número de copias (e.g. ~103 copies)
Señ
al
(F
luo
resc
enci
a n
orm
aliz
ada)
Tiempo (ciclos de amplificación)
threshold = umbral de deteccción
Curva de Amplificación QPCR
Alto número de copias (e.g. ~108 copies)
Fase inical lag No detecta señal
Fase log Señal ~ el doble cada ciclo
Fase plateau Reacción es lenta & se detiene cuando se tiene un reactivo limitante.
Punto Final (no cuantitativo)
Bajo
= mas copias, amplifica
antes
Alto CT
= menos copias,
amplifica despues
CT
CT = threshold cycle (Numero de ciclo en donde la señal de amplificación cruza el umbral de detección.
Curva de amplificación QPCR
Dos tipos de formatos de Detección
Sondas secuencia específica: Hibridación e hidrólisis
HybProbes Dual-labeled probes (sondas TaqMan)
Detección inespecifica
utilizando colorantes que se intercalan en el DNA de doble cadena.
Solo hay un colorante
Es más económico
SYBR® Green
Formato SYBR Green
SYBR Green
ss DNA
Taq polymerase
ds DNA
Sondas de Hibridación e Hidrólisis: Fundamento
FRET: Fluorescence-Resonance-Energy-Transfer Definition
Transferencia de energia a un fluoroforo excitado (donador) a otro fluoróforo (aceptor) o bien a una molecula no fluorescente
Dark Quencher Fluorophore
heat
Fluorophore 2 Fluorophore 1
input radiation
output radiation
input radiation
PCR en Tiempo Real
1. Sondas de Hibridación
Incrementa la fluorescencia en cada ciclo
La señal depende unicamente de la hibridación
1. Sondas de Hidrólisis
Incrementa la fluorescencia en
cada ciclo La señal depende unicamente de la
hibridación
Hybridization Probes Molecular Beacons
Chemistry Considerations
There are many chemistry types TaqMan, Eclipse, Beacons, Adjacent dual (FRET) Probes, LUX primers, Scorpion probes, etc
There are many chemistry variants MGBs, LNAs, “superbases”, inosine substitutions, etc
Trend: the chemistry provider designs the assay
Providers understand idiosyncrasies of their chemistry They usually have proprietary design software They usually design for free if you purchase
Recommended you always consult chemistry provider about assay design!
Ventajas de la PCR en Tiempo Real
Riesgo de contaminación Mínimo
Quantificación
Reduce los procesos de estandarización y variaciones de operarios
Ideal para diagnóstico clínico
Rapidez
Mayor sensibilidad
Mayor especificidad
Mayor precisión
Ethidium-Gel
detection
Cuantifiación absoluta
– Se realiza cuando se desea conocer los niveles de expresión de un
gen indicados como un número absoluto (copias, concentración, pg
etc), en NUMEROS.
– Para la cuantifiación se requiere realizar una curva estándar con al
menos cinco estándares por triplicado
– Los estándares deben representar preferentemente diluciones
1:10, para esperar una diferencia de Ct de 3.162 entre cada dilución
– Importante: La calidad de la cuantifciación absoluta esta dada por:
• Pipeteo correcto
• Preparación de los estándares
• Buena conservación de los estándaes
amount
5,000 50,000 500,000 5,000,000 50,000,000 500,000,000
CT
amount
Muestra
desconocida
determinada
por la curva
estándar
5,000 50,000 500,000
5,000,000
50,000,000 500,000,000
CT
Cantidad
1. Graficar CT vs log concentración 2. Utilizar la gráfica que mejor se ajuste
3. Calcular la eficiencia a partir de la gráfica La ecuación es la siguiente, donde m es la penciente E= 10(-1/m) – 1 E=1=100%.
4. Eficiencia de correlación (R2) correlación entre el valor teórico y e valor real. La correlación debe ser lo más cercana a 1
Cuantificación absoluta y eficiencia de reacción
Panel Transplante
•Infecciones Diversas después del Transplante
– infecciones Virales
– infecciones Fúngicas
.Herpes virus
CMV
EBV
HSV 1/2
VZV
Aspergillus
Panel Encefalitis
.Panel Encefalitis
Patógenos virales más comunes
HSV-1/2
Otros virus
Influenza
CMV , EBV (Herpes)
Patógenos Bacteriales
Mycobacterium tuberculosis
Panel Infecciones Intra-Hospitalarias
MRSA S. aureus C. difficile
Panel Infecciones Salud de la Mujer
C. trachomatis N. gonorrhoeae GBS
Panel Sangre
HIV
Virus Hepatitis
HBV
HCV
PHC therascreen - Fast results for timely treatment decisions
Personalized Health Care
Companion diagnostics
Patients
Payers
Providers Pharma companies
Physicians
All stakeholders benefit
Industry-leading portfolio of co-development projects
Project Partner Indication Biomarker Status
Eribitux (cetuximab)
Colorectal cancer
KRAS U.S. regulatory approval in July 2012
Vectibix (panitumumab)
Colorectal cancer
KRAS U.S. regulatory approval in June 2014
PF-00299804 (dacomitinib)
Lung cancer (NSCLC)
KRAS In development
Iressa (gefitnib)
AstraZeneca Lung cancer (NSCLC)
EGFR CE-IVD kit in Europe and other non-U.S. markets
Gilotrif (afatinib)
Lung cancer (NSCLC)
EGFR U.S. regulatory approval in July 2013
Early-stage compound Blood cancer JAK2 In development
Various projects
Not disclosed Various In development
Not disclosed Confidential oncology Confidential In development
Not disclosed Confidential Non-oncology Confidential In development
Therascreen portfolio to address cancer biomarkers
•Disease Areas
Brain
MGMT
IDH1 & IDH2
(BRAF)
Colorectal
KRAS
NRAS
BRAF
PIK3CA
UGT1A1
Melanoma
BRAF
NRAS
* Under development
NSCLC
EGFR
ALK
KRAS*
(NRAS)
(BRAF)
GIST
KIT & PDGFRA
(BRAF)
www.mycancergenome.org
Leukemia (ipsogen)
JAK2 V617F
BCR-ABL1
PML-RARA
WT1
NPM1
….
Non-small cell lung carcinoma (NSCLC)
Drug efficacy correlates with EGFR mutation status
IPASS (IRESSA Pan-ASia Study): randomised, parallel-group study to assess the efficacy, safety and
tolerability of gefitinib (novel drug) versus carboplatin/paclitaxel (standard therapy) as first line treatment in a
clinically selected patient population from Asia.
Gefitinib or Carboplatin–Paclitaxel in Pulmonary Adenocarcinoma. TS. Mok, Y-L. Wu, S. Thongprasert, C-H. Yang, D-T. Chu, N, Saijo, et al., NEJM, Sep 2009; 361: 947-957.
IPASS Study
Afatinib versus pemetrexed/cisplatin as a first-line treatment in advanced lung cancer harbouring EGFR-activating mutations. Yang JC-H, et al. ASCO 2012: Abstract LBA7500, LUX-Lung 3:
Lux Lung 3 trial
Non-small cell lung carcinoma (NSCLC)
Drug efficacy correlates with EGFR mutation status
Afatinib
Pemetrexed/cisplatin
Only patients EGFR mutation positive included in the study
tested with Qiagen kit Patients benefit from Afatinib (Gilotrif) Kit and drug FDA approved
Technology Landscape: Advantages therascreen
PCR:
Real-time
Detection
PCR
Clean-up
Pyro-
Sequencing
Reaction
PCR
Heat
Destroy
Polym
Invader
Reaction
Detection
via
Plate Read
therascreen RGQ & Pyro PCR/Invader
PCR
Clean-up
Sequencing
Reaction
Clean-up
PCR/Sequencing
Capillary
Sequencing
= Transfer product/ add reagent/ open tube
PCR
Clean-up
Sequencing
Reaction
Clean-up
SNaPshotTM
Capillary
Sequencing
PCR
PCR
Clean-up
Sequencing
Reaction
Nested PCR/ Seq
Clean-up
Capillary
Sequencing
1
Steps
2
3
4
5
6
PCR:
Real-time
Detection
PCR
Clean-up
Pyro-
Sequencing
Reaction
PCR
Clean-up
Pyro-
Sequencing
Reaction
PCR
Heat
Destroy
Polym
Invader
Reaction
Detection
via
Plate Read
therascreen RGQ & Pyro PCR/Invader
PCR
Clean-up
Sequencing
Reaction
Clean-up
PCR/Sequencing
Capillary
Sequencing
= Transfer product/ add reagent/ open tube= Transfer product/ add reagent/ open tube
PCR
Clean-up
Sequencing
Reaction
Clean-up
SNaPshotTM
Capillary
Sequencing
PCR
PCR
Clean-up
Sequencing
Reaction
Nested PCR/ Seq
Clean-up
Capillary
Sequencing
1
Steps
2
3
4
5
6
We offer the fastest, easiest to use, most sensitive and CE-IVD approved methods and kits
ARMS Scorpions qPCR
Highest sensitivity on the
market
Selective mutation detection
Ease of use
One-step procedure
Pyrosequencing
Sequence information
Complex mutation testing
with simple data interpretation
Small amount of starting material
QIAGEN solutions for molecular diagnostic
KRAS
EGFR
BRAF
And more…
Mutation testing
Criteria for choosing PCR or Pyro kits
Parameter PCR Pyro
What type of information is needed Restricted to mutations that are
clinically relevant (closed system) Sequencing data (open
system)
Technology preference of the customer PCR Sequencing
Most important criteria of the assay Faster result and minimum handling Detailed report
Report format Results YES/NO Sequencing data
General Lab profile Diagnostic or routine service lab Diagnostic lab with R&D
activities
Experience in interpreting results
clinical/technical background of user Basic skills Experience required
Experience in molecular biology No Yes
therascreen RGQ Kit Portfolio
Sample assessment run
Result interpretation
~ 2h ~ 15 min
RGQ Kit workflow overview
PCR Setup
~ 20min
PCR Setup
82
Mutation detection run
~ 2h ~ 20min
Myeloproliferative neoplasms (MPN) spectrum
Philadelphia
(Ph) -
Philadelphia
(Ph) +
MPN
Polycythemia
vera (PV)
Essential
thrombocythemia (ET)
Primary
myelofibrosis (PMF)
Chronic myelogenous
leukemia (CML)
~ 65%
JAK2 V617F
positive
~ 55%
JAK2 V617F
positive
Sources: Tefferi, A. (2008) Leukemia; Patnaik, M.M. (2009) Leukemia; Tefferi, A. (2010) Leukemia..
~ 96%
JAK2 V617F
positive
ipsogen JAK2 RGQ PCR Kit CE Content
4 dilutions for JAK2 Wild-Type quantification:
• WT-1: 50 copies standard • WT-2: 500 copies standard • WT-3: 5 000 copies standard • WT-4: 50 000 copies standard
4 dilutions for JAK2 V617F quantification:
• MT-1: 50 copies standard • MT-2: 500 copies standard • MT-3: 5 000 copies standard • MT-4: 50 000 copies standard
JAK2 Mutant Control (MTC): genomic DNA harboring 100% JAK2 V617F allele
JAK2 WT Control (WTC): genomic DNA harboring 100% JAK2 wild type allele
JAK2 WT Rx (Primers & Probes reaction mix) - FAM specific for JAK2 Wild-
Type allele - HEX specific for IC
JAK2 MT Rx (Primers & Probes reaction mix) - FAM specific for JAK2 V617F
mutant allele - HEX specific for IC
QIAGEN qPCR master-mix including Internal control (IC)
IVD
Catalog number: 673623
LOD at 0.042% JAK2 V617F
Water for NTC TE buffer for sample dilution
100%
10%
1%
0.1%
0.01%
0.001%
0.0032%
LoD of previous kit : ipsogen BCR-ABL1 Mbcr Kit
LoD of previous kit : ipsogen BCR-ABL1 Mbcr IS-MMR Kit
Limit of detection (LOD) and molecular response
•Improved sensitivity
0.0069%
0.08%
LoD of new ipsogen BCR-ABL1 Mbcr RGQ RT-PCR kit
0.0030% MR4.5
MR5
MR4
MMR
Myeloproliferative Neoplasm (MPN) Spectrum
Philadelphia
(Ph) -
Philadelphia
(Ph) +
MPN
Polycythemia vera (PV)
22% in EU
Essential thrombocythemia
(ET) 33% in EU
Primary myelofibrosis
(PMF) 10% in EU
~ 55-65%
JAK2 V617F
positive
~ 50-60%
JAK2 V617F
positive
~ 97%
JAK2 V617F
positive
Simplified from Tefferi, A., Vardiman, J.W. (2008) Leukemia. 22, 14. Patnaik, M.M., Tefferi, A. (2009) Leukemia. 23, 834. Tefferi, A., et al. (2010) Leukemia. 24, 105. Barbui et al. (2015) Blood Cancer Journal. 5, e337. Incidence and prevalence in the American Journal of Hematology, Vol. 00, No. 00, Month 2014.
>90%
BCR-ABL1 Mbcr
Chronic myelogenous leukemia
(CML) 35% in EU
~ 20-25%
CALR
positive
~ 20-25%
CALR
positive
~ 7%
MPL
positive
~ 3%
MPL
positive
Myeloproliferative Neoplasm (MPN) Spectrum
Philadelphia
(Ph) -
Philadelphia
(Ph) +
MPN
Polycythemia vera (PV)
22% in EU
Essential thrombocythemia
(ET) 33% in EU
Primary myelofibrosis
(PMF) 10% in EU
~ 55-65%
JAK2 V617F
positive
~ 50-60%
JAK2 V617F
positive
~ 97%
JAK2 V617F
positive
>90%
BCR-ABL1 Mbcr
Chronic myelogenous leukemia
(CML) 35% in EU
~ 20-25%
CALR
positive
~ 20-25%
CALR
positive
Epigenetics Definition and Mechanisms
DNA Methylation * Adding of a methyl group on C residues of CpG dinucleotides
* CpG islands are often found in promoter regions * Genes in methylated DNA are silenced
Histone Modification * Acetylation, methylation, and phosphorylation of histones * Histones are associated with DNA as chromatin * Acetylation of histones results in chromatin that is generally transcriptionally active
miRNA * Post-transcriptional control of gene expression * microRNA can bind to 3’-UTR of mRNA and affect translation of mRNA * microRNA downregulate gene expression
Epigenetics Mechanisms
The study of reversible inheritable influence on gene activity that is
not accompanied by a change in the DNA sequence
What Is Micro RNA?
.Endogenously expressed small functional RNAs (~ 21-23 nt)
.Regulate mRNA expression post-transcriptionally • Primarily translation inhibition, but also mRNA
degradation
.Only partial sequence complementarity required • One miRNA can regulate multiple mRNA • One mRNA can be regulated by multiple miRNA
.Another layer of complexity for regulating gene expression
A look back 10 years Unique miRNA signatures are
found in human cancer
– miRNAs located in genomic regions amplified in cancers (e.g. miR-17-92 cluster) can function as oncogenes, whereas miRNAs located in portions of chromosomes deleted in cancers (e.g. miR-15a-miR-16-1 cluster) can function as tumor suppressors.
– Abnormal expression of miRNAs has been found in both solid and hematopoietic tumors.
– miRNA expression fingerprints correlate with clinical and biological characteristics of tumors, including tissue type, differentiation, aggression and response to therapy.
In the last 10 years, a substantial number of studies and reviews have associated the
presence of various miRNAs with cell proliferation, resistance to apoptosis, invasiveness, and differentiation in cancer cells.
Potential events leading to disruption of ‘normal’ miRNA : target interaction in disease
microRNA Gene
mature miRNA
Pre-miRNA
Drosha-DGCR8
Exportin
DICER-TRBP
Pri-miRNA
Ago
Target Transcript
miRNP
Altered Transcription
Methylation
Histone Modification
Transcription Factor
Drosha Processing
Genomic Instability
Amplification/Deletion
Translocation
Insertional Mutagenesis
Loss of miRNA Binding Site in target
SNP or Mutation
Alternative Splicing
Loss/Change of 3’-UTR
Dicer Processing
miRNA in Blood RBC, WBC, platelets, CTC, “other cells”, extracellular?
What is blood? RBC, WBC, platelets, other cells (e.g. circulating tumor cells) Serum (post clotting) Plasma (no clotting)
High levels of nucleases present in plasma
Freely circulating RNA should be rapidly degraded
Surprisingly, stabile miRNA can be detected in serum and plasma
1) Valadi, H., et.al.,(2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat Cell Biol 9:654-659
2) Hunter MP et. al., (2008) Detection of microRNA Expression in Human Peripheral Blood Microvesicles, PLoS ONE 3:e3694 3) Kosaka, N et. al (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells, J Biol Chem 285: 17442-17452 4) Arroyo, JD et. al., (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma,
Proc. Natl. Acad. Sci 108: 5003-5008 5) Vickers, KC., et. al., (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol
13:423 6) Wang K, Zhang S, Weber J, Baxter D, Galas DJ.(2010) Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic
Acids Res. 2010 Nov 1;38(20):7248-59.
Exosomes & micro vesicles1,2,3
miRNA
Ago
Ago-2-miRNA complexes4
HDL mediated miRNA transport5
Other “protective” protein6
Stable miRNA in circulation ~2007 Contained in exosomes, microvesicles, other: An evolving story
Extracellular miRNAs are in all body fluids
Weber et al., (2010) Clinical Chem. 56: 1733-1741 miRNeasy, miScript PCR System
Microbiome: From identification to characterization
Humans or Superorganisms? •Cellular composition of the organism
97
Human
Microbiota
Estimations of the number of microbial cells that live in and on the human body, human cells are outnumbered by a factor of 10. Nomenclature: Microbiota are the microbes that live in a specific location, e.g. the human body, the gut, soil, etc. Metagenomics is the study of the collection of genomes derived from a specific sample or community. Microbes are microscopic organisms that can be either single or multicellular. For the purposes of this webinar, we will use microbiome to describe the collective genomes of the microbiota that inhabit a specific location.
Microbiota composition •Microorganisms cluster by body site
98
•Cataloguing efforts by the NIH Human microbiome project suggest:
•~10,000 organisms live with us
•~ 8 ×106 genes in this “second genome”
•Identifying microbiota in healthy individuals revealed:
•Different body sites have unique communities
•Race, Age, Gender, Weight or Ethnicity have no effect
Complexity and function of genomic content
•Function of microbiome enables individual survival
99
•Each organism has developed genetic content for its own survival in a specific environment.
•Metabolism tuned to local nutrient sources
•Virulence factors for stable colonization
•Antibiotic resistance genes to metabolize toxins
Current Methods for Microbial Analysis
•Identifying microbes and their genes
• Culture – Nutrients – Antibiotics
• 16S rRNA gene clone library construction – Pan 16S rRNA PCR amplification cloned sequenced
• Microarray
• Next generation sequencing – 16S rRNA sequencing – Whole genome sequencing
• MALDI
• qPCR – Target dependent (16S rRNA or gene)
100
16S rRNA gene as a phylogenetic marker for
bacterial ID
– Classification from the variable sequences
16s rRNA sequence similarity
95% genus level, 97% species level, 99% strain level
101
16S rRNA gene
- Conserved region - Variable region
Association of the human microbiota and disease
– Gut
• Intestinal infections
• Obesity
• Inflammatory Bowel Disease
– Airway
• Pneumonia and other respiratory infections
• Chronic Obstructive Pulmonary Disease
• Cystic Fibrosis
– Urogenital
• Bacterial Vaginosis
• Urinary Tract Infections
• Sexually Transmitted Disease
– Blood
• Sepsis/Blood-stream infections
– Cancer
– Heart disease
– Neurological disorders
– Oral
• Periodontitis
• Gingivitis
102
Correlate qPCR assay performance with NGS results
•Profiles of vaginal flora by qPCR and whole genome sequencing
103
Microbial DNA qPCR Arrays Protocol
•Identification or Profiling
104
Antibiotic resistance gene reservoirs in the body
•Screening of the gut for presence of antibiotic resistance genes
105
500 ng of genomic DNA from stool samples originating from five healthy adults were analyzed for presence of antibiotic resistance genes. 87 unique antibiotic resistance genes were tested for by qPCR. Positive (+) / negative (blank) result for each antibiotic resistance gene was determined using a threshold cycle cutoff.
Cervical flora: Gardnerella vaginalis positive vs. BV negative
106
Cervical flora: Gardnerella vaginalis positive vs. BV negative
In samples with high Gardnerella vaginalis abundance, there was an increase in co-occurrence of BV-associated microorganisms and decrease in abundance of the normal flora, Lactobacillus crispatus.
107
Antibiotic resistance genes in our food supply?
108
Mejorando vidas mediante el impacto clinico de NGS Presentando el sistema de NGS GeneReader
• QIAGEN NGS Basics
110
NGS 101: The Basics NGS Basics Content Responsibility Additional Materials Time
Introduction to NGS Background of NGS GPM NOW 1 training Slides
NGS Market Sequencers/ Companies / Opportunities
GPM
NGS Applications and Experiments at QIAGEN
WGS, WES, Targeted Panel(DNA), transcriptome, Targeted RNA, miRNA, liquid biopsy,
GPM
Basic Overview of Portfolio
DNA library prep GPM Portfolio flyer/ training deck
Selling NGS: Questions /Selection Guide/Key Terms
GPM+ specialist or regional marketing
Selection Guide
Feedback
What does NGS do? •Decipher the nucleotide composition in a DNA sample
CONFIDENTIAL, DO NOT DISTRIBUTE.
DNA sample
ACCTAGTCATGAGTCATGCAT
Nucleotides
Transform the DNA sample into a collection of nucleotides
What makes a sequencing method “Next Generation”?
1
1
3
1 Cluster (DNA spot) = 1 read
What are we talking about?
NOW training-NGS Basics 114
1st Generation Sequencing Sequence many identical
molecules
Using any old sequencing chemistry
In a tube
2nd Generation Sequencing (NGS) Sequence many identical
molecules (usually)
Using a reversible, step-wise sequencing chemistry
Immobilized on a surface
General NGS Workflow
NOW training-NGS Basics
Data Analysis &
Interpretation
2nd generation Sequencing
Sample Extraction
Library Preparation
What does NGS data look like?
PowerPoint Style Guide 116
Short answer: Unintelligible times a billion
(seriously, these files are huge and will make your computer cry if you try to open them)
How do you sequence? •You use NGS platforms
CONFIDENTIAL, DO NOT DISTRIBUTE.
gDNA
ACCTAGTCATGAGTCATGCAT
Nucleotides
Ion Torrent NGS platforms
Illumina NGS platforms
Why NGS?
What NGS library prep does
1. Create DNA fragments
2. Add platform-specific sequences to every fragment
3. Amplify library molecules for QC and sequencing (if necessary)
“Stuff”
Library Preparation
Data Analysis &
Interpretation
2nd generation Sequencing
Sample Extraction
NGS Library? •NGS platforms sequence libraries
CONFIDENTIAL, DO NOT DISTRIBUTE.
DNA
ACCTAGTCATGAGTCATGCAT
Nucleotides
Library
DNA needs to be turned into libraries before it can be sequenced Library = millions of small DNA fragments
What is a library? •Pieces of DNA to be sequenced flanked by adapters
CONFIDENTIAL, DO NOT DISTRIBUTE.
DNA
ACCTAGTCATGAGTCATGCAT
Nucleotides
DNA to be sequenced
Adapters
How do I generate the small pieces of DNA?
CONFIDENTIAL, DO NOT DISTRIBUTE.
DNA
ACCTAGTCATGAGTCATGCAT
Nucleotides Library
Small pieces of DNA
Shearing – physically breaking DNA into small pieces
Fragmentation – enzymatically digesting DNA into small pieces
Multiplex PCR – use primers to generate small amplicons
Enables targeted sequencing using panels
What do adapters do?
CONFIDENTIAL, DO NOT DISTRIBUTE.
•Multiplexed Sequencing: Multiple samples sharing one sequencing reaction to reduce per-sample costs and control the amount of data generated per sample.
CONFIDENTIAL, DO NOT DISTRIBUTE.
124
What the adapters do? : Add barcodes for multiplexing
Barcode 1
Each 96-plex adapter includes two 8-base barcodes 8 Barcode 1’s x 12 Barcode 2’s = 96 combinations
Pool barcoded libraries
single multiplexed
NGS run
Demultiplexing
fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq
Separate fastq data files for each sample
Adapter Ligation Point
How do you sequence? •NGS platforms are not enough. You need to use a flow cell or chip on NGS platforms
CONFIDENTIAL, DO NOT DISTRIBUTE.
gDNA
ACCTAGTCATGAGTCATGCAT
Nucleotides
GeneReader workflow uses flow cells
Ion Torrent NGS platforms use
beads on chips
Illumina NGS platforms use flow cells
Flow cells and chips are important because the entire sequencing run takes place on their surfaces
You generated reads. Now what? •Reads have to be analyzed through data analysis (bioinformatics) pipelines
CONFIDENTIAL, DO NOT DISTRIBUTE.
Variants (mutations)
Read 1000000
Read 1000
Read 1
What is the principle of data analysis? •Compare DNA sequences in your sample to the DNA sequence of the reference genome
CONFIDENTIAL, DO NOT DISTRIBUTE.
ACAGTTAAGCCTGAACTAGACTAGGATCGTCCTAGATAGTCTCGATAGCTCGATATC
AACTAGACTAGGATCGTCCTAGATAGTCTCG
Reference sequence
Aligned reads
AACTAGACTAGGATCGTCCTACATAGTCTCG
AACTAGACTAGGATCGTCCTACATAGTCTCG
GATCGTCCTAGATAGTCTCGATAGCTCGAT
GATCGTCCTAGATAGTCTCGATAGCTCGAT
GATCGTCCTAGATAGTCTCGATAGCTCGAT
There is a nucleotide change from G to C at this position in my sample compared to the
reference genome (which is considered a normal genome)
One can take an additional step to interpret what those genetic changes
mean (data interpretation)
Putting it all together
CONFIDENTIAL, DO NOT DISTRIBUTE.
DNA purification
Data Analysis &
Interpretation
Library preparation
Sequencing Sample
Collection and Stabilization
Tradeoff Between Depth and Coverage
NOW training-NGS Basics
All Bases
1. Whole Genome
2. Hybrid Capture
(Exome)
mRNA-Seq (Transcriptome)
3. DNA Targeted
Panels
Affordable Sequence Depth
30X WGS
100X Exome
1000X+ Amplicon Seq RNA
Targeted Panels
4. RNA
1X Low-pass (SC)
Selected NGS Applications
•Large fraction of genome = greater costs, but greater ability to compare samples or patients across studies •Small targeted panels = easier and less expensive to complete the project, but limited ability to follow up on new hypotheses or discover new associations
NOW training-NGS Basics
Whole Genome Exome Sequencing DNA Amplicon Panels RNA-Seq (Transcriptome)
Free circulating nucleic acids
RNA and DNA from dead cells shed into the bloodstream, can contain cancer-related mutations.
What is Liquid Biopsy? •A minimal invasive technology for detecting signs of cancer and other diseases
Tissue samples
FFPE tissue samples of tumor extracted from the patient’s body through an invasive procedure.
Comprehensive portfolio offering proven ways to access nucleic acids
Exosomes
Tiny microvesicles found in body fluids that transport RNA between cells.
Circulating Tumor Cells
Tumor cells shed from a tumor into the bloodstream carrying genetic information.
Oncología: Líder en Medicina Personalizada
Launch Event GeneReader Mexico City 132
GeneReader NGS First complete Sample to Insight NGS solution, expansion to companion diagnostics
Lung Breast Colon Hematology
ipsogen Widest range of molecular tests for blood cancer
therascreen No. 1 portfolio of solid tumor companion diagnostics
Cervical
digene The proven gold standard for cervical cancer detection
Providing critical diagnostic solutions across clinical disease progression
insights along cancer care continuum Industry-leading pharma collaborations
Disease unknown
Hybrid Capture (HC2) Population screening
Disease suspected
Next-generation sequencing Metastatic cancers
Initial diagnosis
Translational research
Disease diagnosed
Targeted PCR / NGS Resistance detection
Treatment modification
Companion diagnostics
Disease monitoring
Targeted PCR / NGS Refraction
Recurrence
La promesa en NGS Consolida las pruebas para incrementar la eficiencia en su laboratorio
Limitaciones de los ensayos moleculares tradicionales:
• Múltiples pruebas para detectar diferentes variantes y tipos de las mismas
• Riesgo de no tener suficiente material
• Incrementar el tiempo de respuesta y hacer eficientes los rescursos
NGS resuelve estos problemas
¿Qué evita a los laboratorios de convertir en realidad la promesa de NGS?
Altos costos
Flujos de trabajo fragmentados
Análisis e Interpretación de datos
¿Qué evita a los laboratorios de convertir en realidad la promesa de NGS?
Seamless Bioinformatics
Simple workflow Cost-effective
¿Qué hace diferente al sistema de NGS GeneReader?
La primera solución completa a nivel mundial que cubre de la
muestra al detalla en el campo de NGS
Diseñada para entregar información con impacto clínico
“The QIAGEN GeneReader NGS System is
the first truly integrated solution that is
focused on delivering critical insights with
maximal efficiency and minimal adoption
hurdles.”
—Scott Steelman, PhD, Associate Director of Technology Labs
Launch Event GeneReader Mexico City
“If you combine different
products and technologies from different
companies, you never
know how well they will fit and function
together.”
“People tend to largely underestimate the
true cost of running NGS, not considering
the cost of an often lengthy and pricy
optimization process. With the
GeneReader NGS System, I expect to see
a major advantage from a fully integrated
workflow.”
CAP Today, March 2017
1. Evitar el dolor de sincronizar equipos accesorios de otros proveedodres
2. Ahorrar el tiempo de recursos especializados para desarrollar y optimizar el análisis bioinformático
3. Proveer una solución verdadera con un secuenciador previamente validado en su flujo de trabajo
Beneficios de la integración de los flujos de trabajo
1
3
9 Integrado
Actionable
Flexible
Accesible
Cofiable
Not a Sequencer---but a System
L
a
u
n
c
h
E
v
e
n
t
G
e
n
e
R
e
a
d
e
r
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e
x
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o
C
i
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y
1
4
0
Nucleic Acid Extraction
Target Enrichment
Library Preparation
Next-Generation Sequencing
Data Analysis
Interpretation
QIAxcel QIAxcel RGQ
QC
GeneRead Assistant (iPad and App included)
Support
Data Management
GeneRead Link and LIMS GeneRead Databank
QIAcube QIAcube RGQ GeneRead QIAcube
GeneReader QCI-Analyze QCI-Interpret
Automation
El panel que aporta más información de impacto clínico
• KRAS
• NRAS
• KIT
• BRAF
• PDGFRA
• ALK
• EGFR
• ERBB2
• PIK3CA
• ERBB3
• ESR1
• RAF1
Enfocado en tumores sólidos con:
• La prevalencia más alta
• Necesidad más grande de detección
• Variantes asociadas a información clínica
QIAGEN Knowledge Base
Lung
Breast
Ovarian Colorectal
Melanoma
12 genes
Integrado
Actionable
Flexible
Accesible
Confiable
QIAact BRCA 1/2 Panel: no solo para el cancer de mama hereditarios
•
Tipo de Cancer Tipos de mutaciones Guías NCCN/SGO
recomiendan Clinical Actionability
• Mama • Ovario
• Germinales • Somaticas
• Toda mujer en edad reproductiva
• Pacientes con alto riesgo de cancer mamario
• Inhibuidor PARP
• Protocolos Activos para mutaciones somaticas
Detección de mutaciones en Sangre, FFPE y múltiples tipos de canceres
QIAact Lung MB Panel – need for test consolidation
•
Cambios geneticos complejos , por ejemplo:
Mutación Puntual: EGFR T790M
Deleción grande MET: exon 14
CNV: ALK
Fusiones: RET, ROS1
Pruebas actualmente en uso: • PCR • IHC • FISH • Arreglos
Detección en un sólo ensayo que proporciones todos estos resultados
QCIA – Intuitive and flexible variant calling and visualization
•Fully automated alignment, quality control, variant calling
El QIAact panel surge de la base de conocimiento generada por QIAGEN
Base de conocimientos de QIAGEN
• Comprende la infromación generada por la empresa líder del segmento
• Compila multiples fuentes publicas y privadas
• Está depurada y continuamente actualizada
El diseño del panel está basado en :
• Medicamentos disponibles
• Protocolos Clinicos
• Guías Profesionales
Integrado
Actionable Flexible
Accesible
Confiable
Un panel diseñado para aportar información con impacto clínico relevante
QCII – Intuitive and transparent variant interpretation
•Designed, built and validated in collaboration with clinical labs
Customizable Reporting
ability to capture /persist internal annotations, integration into LIMS and EMR systems
Comprehensive Curation of Clinical Evidence
reported cases, drug labels, professional guidelines
Decision Support Software classify variants, identify treatment options, and
geographical clinical-trial matching
Treatment and Trial Identification
based upon levels of evidence, expected drug response, prognosis and lab defined rules
QIAact Panel Portfolio
Actionable Insights Tumor Panel Lung DNA & Fusions
Panel
Myeloid Leukemia Panel
BRCA 1/2 Panel
Custom Panel
Based on QIAGEN knowledge base, enabling maximum actionability Verified from sample to insight
No Invasive Pre-Nata Test (NIPT)-Natera
Now Now Now
November with UMIs
Q4
Now
Q3
QIAGEN es el líder global en calidad y servicio
1
4
8
>35 offices
worldwide
>550 service
specialists
>15,000 installations
>4,000 employees
>500,000 customers
Integrado
Actionable
Flexible
Accesible
Confiable
Flexibilidad que se ajusta a sus necesidades
•Un batch se compone de 1 a 10 pacientes; GeneReader puede procesar hasta 4 Flow Cells
new new
GeneGlobe inside
1-10 pacientes 1 Amplificación
clonal 1 Flow Cell 1-10 Reportes
Patient/ clinical
data
Patient/ clinical
data
Patient/ clinical
data
Patient/ clinical
data
Patient/ clinical
data
Patient/ clinical
data
Patient/ clinical
data
Patient/ clinical
data
Patient/ clinical
data Patient/ clinical
data
40 pacientes 4 Amp Clonal 4 Flow Cell 40 Reportes
120 pacientes 12 Amp Clonal 12 Flow Cell 120 Reportes
Por corrida
Por semana
Por Flow Cell
Molecular Oncohemathology Workflows: From Sample to Insight
Jacobo Zuñiga Castillo Ph.D. Regional Marketing Manager
QIAGEN Mexico, Central America and Caribbean