Presentación de PowerPointlabclin2016.pacifico-meetings.com/images/site/Ponencias_LabClin2… ·...
Transcript of Presentación de PowerPointlabclin2016.pacifico-meetings.com/images/site/Ponencias_LabClin2… ·...
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Aplicaciones de la secuenciación masiva
en el manejo de la hepatitis C
Josep Quer
Liver Unit. Hepatitis Virus. Vall d’Hebron Institut of Research (VHIR). Hospital Universitari Vall d’Hebron (HUVH). UniversitatAutònoma de Barcelona.
Ciber Enfermedades Hepáticas y Digestivas (Ciberehd) del Instituto de Salud Carlos III. Madrid.
Sociedad Española de Virologia (SEV).
Congreso Nacional del Laboratorio Clínico 2016
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MASSIVE SEQUENCING or NEXT GENERATION SEQUENCING (NGS) or DEEP SEQUENCING orMASSIVE PARALLEL SEQUENCING
Sequencing of 100.000 to Millions (1,000,000- 43 billion) of DNA fragments (50-1000 bases each) per instrument run at the same time (in parallel).
MASSIVE SEQUENCING PLATFORMS
Platforms 2016 (size of reads):
Illumina (Solexa) sequencing: 300-500nt
Roche 454 sequencing (Roche): 400-1000nt
Ion torrent:Proton/PGM sequencing (Thermo Fisher Scientific): ~200nt
SOLiD sequencing (Applied Biosystems): 50-60nt
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Unknown sequence
To fragment DNA by nebulitzationor other method.
Known sequence
Use labelledprimers (PCR, RT-PCR)
+Ligation oligon.
Known end
SEQUENCING using any NGS platform
SEQUENCING A GENETIC MATERIAL (DNA or RNA)
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1950 1960 1970 1980 2009
1953 discoveryDouble helixstructure of DNARosalind ElsieFranklin,Watsony Crick
1977Sanger
sequencingmethod
1983PCR
Kary Mullis
1990 2000
1990HGPStart
13 years(3Gb)
Human GenomeProject (HGP). Public
Consortium
2003HGPCompleted
NGS
5 days (5Gb= 1,5 genomes)
2007
NGS
James Watson´sgenome1 month(3Gb)
Classical Sanger Sequencing
History of Human Genome Sequencing.From SANGER to NGS
2016: One humangenome(3Gb=3000Mb) can be sequenced within a single day
2016 ---- 1000$ ¿?
BIG SEQUENCING PROJECTS
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SOME BIG SEQUENCING PROJECTS
http://www.1000genomes.org
TCGA project
April 2016
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WHAT ABOUT HCV!!!
Aplicaciones de la secuenciación masiva
en el manejo de la hepatitis C
Congreso Nacional del Laboratorio Clínico 2016
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QUASIESPECIES
Martell M et al. (HCV) J.Virol. 1992; 66(5):3225-3229Holland JJ et al. Science 1982; 215(4540):1577-1585 / Domingo E & Holland JJ. Evolutionary biology of viruses. 1994 Vignuzzi Nature 2006; 439:344-348 /Vignuzzi M, et al. Nature 2005.
Estimated Rate of Mutation HCV
10-3- 10-4
substitutions/nt/replication cycle
High level of replication of HCV
HCV 1012particles/day
QUASISPECIES NATURE OF HCV
CONSENSUS
MUTANT
ESPECTRA
RNA polimerases LACK OF PROOFREADING MECHANISMS.=High mutation rates 10-3 - 10-5
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Resistance-Associated amino acids Substitutions (RAS) to DAAs
Escape from VACCINATION
Induction of Peripheral tolerance= Chronicity
Cured patients can be reinfected
Resistant mutants can be transmitted (Franco S, et al. Gastroenterology. 2014 Sep;147(3):599-601)
...
HCV
HCV QUASISPECIES.HIGH CAPABILITY TO GENERATE MUTATIONS:
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QUASISPECIES. POPULATION OF SEQUENCES
CONSENSUS
1010-1012
SANGER SEQUENCING (POPULATION SEQUENCING)
= 1 seq (CONSENSUS)
MASSIVE PARALLEL
SEQUENCING (NGS)
= thousand of seqs
BEST TOOL to STUDY the
composition of thequasispecies
1
Congreso Nacional del Laboratorio Clínico 2016
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HCV is NOT a single SEQUENCE, but a POPULATION!
HCV
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SEQUENCE v HAPLOTYPE
10 SEQUENCES from an NGS run
3 HAPLOTYPES(=Different sequences)
6reads
60%30%10%3reads
1read
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Simmonds P & Smith D. Chapter 43: Evolution of hepatitis viruses pp575-586 in Viral Hepatitis. 4th ed. Ed.Thomas,Lok, Locarnini & Zuckerman. John Wiley & sons. Oxford 2014.Simmonds et al. Hepatology 2005
HIV
October 2016 HCV
Genotypes 7
Subtypes 67
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Differences between the three virus that cause the most important human chronic infections.
HCV
DNA ARCHIVENo ARCHIVE
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Insights from deep sequencing. Naturally presence of an Antiviral Resistance Mutant
Domingo, E. Virus as Populations. Academic Press, Elsevier, Amsterdam, 2016
Esteban Domingo’s courtesy
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15HCV
DNA ARCHIVEResistance mutations may
be archived
No ARCHIVE.Persistance of a Resistance
mutant depends on itsFITNESS
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16HCV
1.- FREQUENCY at which an HCV ANTIVIRAL RESISTANCE MUTANT is observed depends of its
FITNESS!!!
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17HCV
FITNESS MEASURES for different SUBSTITUTIONS. Studies in subgenomic replicon.
1.00
0.75
0.25
0
0.50
Fitn
ess
(%)
Donaldson EF et al. Hepatology; 2015; 61:56-65
S282T is the main RAS for SOFOSBUVIR (NS5Bi)
Le Pogam et al JVI 2006Le Pogam et al JID 2010
RAS= Resistance-Associated amino acid Substitution
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Domingo, E. Virus as Populations. Academic Press, Elsevier, Amsterdam, 2016
Esteban Domingo’s courtesy
Insights from deep sequencing. RAS mutations
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19HCV
2.- COMPENSATORY MUTATIONS IN THE SAME GENOME CAN RETRIEVE LOST FITNESS
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Domingo, E. Virus as Populations. Academic Press, Elsevier, Amsterdam, 2016
Esteban Domingo’s courtesy
Insights from deep sequencing. RAS mutationsCongreso Nacional del Laboratorio Clínico 2016
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NS3 Boceprevir Telaprevir Simeprevir Paritaprevir
1a 14m 10.6m 8.3m 6m (40% of patients)12m (9% of patients)
1b 12.5m 0.9m 5.5m
Sarrazin C. J.Hepatol. 2016;64:486-504
NS5A All inhibitors
Long persistance After 1-2 years 85% of patientsNS5A RAS still persist.
MEDIAN TIME TO LOSS OF DETECTABILITY of RAS bypopulation sequencing (Sanger) in months:
NS5B Dasabuvir
Some RAS (M414T, S556G)
S282T*(alone)
6m (75% of patients)12m (57% of patients)7m (in a subject, <1% of sequences with S282T at 7m)
*
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22HCV
3.- RAS mutations BEFORE STARTING TREATMENT may have different meaning than RAS at FAILURE
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3‘UTR5‘NCR
ESTRUCTURAL NO ESTRUCTURAL
PROTEASA Inhibitor (PI)-PREVIR
NS5A Inhibitor (NS5AI)-ASVIR
NS5B Nucloes(t)ídic Inhibitor (Nucs
o NI)-BUVIR
NS5B Non-Nucloes(t)ídic Inhibitor (Non-
Nucs o NNI)-BUVIRSIMEPREVIR (SMV)
PARITAPREVIR/rMK-5172 (Grazoprevir-GRZ) ABT-493 (Glecaprevir)GS-9857 (Voxilaprevir)
DACLATASVIR (DCV)LEDIPASVIR (LEDOMBITASVIRMK-8742 (Elbasvir-EBR)GS-5816 (Velpatasvir-VEL)ABT-530 (Pibrentasvir)
SOFOSBUVIR (SOF)MK3682AL-335MIV-802
DASABUVIR
How to treat in 2016?
AT FAILURE, RESISTANCE SUBSTITUTIONS (RAS) are selected, and can be CROSS-RESISTANT to other INHIBITORS of the same family!!!
In real life: 2-10% of TREATMENTS are FAILING
SPAIN: 53252 treated patients (August 2016)51200 cured = 2052 failures
HARVONI: LEDIPASVIR+SOFOSBUVIRVIEKIRAX: OMBITASVIR-PARITAPREVIR-ritonavir + EXVIERA: DASABUVIREPCLUSA: VELPATASVIR+ SOFOSBUVIRZEPATIER: GRAZOPREVIR + ELBASVIR
EXPECTATIONS FOR THE PATIENTS AND THE PHYSICIAN ARE VERY HIGH!!!
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CUSTOMIZED TREATMENTS. NEED OBJECTIVE DATAFor PERSONALIZING TREATMENTS and
To ELIMINATE THE VIRUS AT THE FIRST TREAMENT
VIRUSPATIENTCOMBINATION OF INHIBITORS (DAAs)
VIRUS:SUBTYPEMIXED INFECTIONSRESISTANCE MUTATIONS
PATIENT:FIBROSIS DEGREETREATMENT-EXPERIENCEDDRUG INTERACTIONS
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2016
1.- Test for 1a/1b, 2-6 must be accurate.2.- No recommendations are done for non-1a/1bsubtypes specially since no commercial tests forsubtyping are available, and lack of information.
GENOTYPING/SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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EASL guidelines 2014EASL guidelines 2015
RAS detectionCongreso Nacional del Laboratorio Clínico 2016
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2016 RAS detectionCongreso Nacional del Laboratorio Clínico 2016
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2016 RAS detectionCongreso Nacional del Laboratorio Clínico 2016
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RAS detection
2017
¿?
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CUSTOMIZED TREATMENTS. NEED OBJECTIVE DATAFor PERSONALIZING TREATMENTS and
To ELIMINATE THE VIRUS AT THE FIRST TREAMENT
VIRUSPATIENTCOMBINATION OF INHIBITORS (DAAs)
VIRUS:SUBTYPEMIXED INFECTIONSRESISTANCE MUTATIONS
PATIENT:FIBROSIS DEGREETREATMENT-EXPERIENCEDDRUG INTERACTIONS
MASSIVE PARALLEL SEQUENCING
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454 / GS-Junior100.000 sequences(=reads) 500-800nts
HIGH-RESOLUTION HCV SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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SPEC
IFIC
MO
DU
LEU
NIV
ERSA
L M
OD
ULE
HCV genome
3‘UTR5‘UTR
STRUCTURAL NONSTRUCTURAL
NS5B (HCV RNA Dependent RNA
Polymerase)
8254 8707
RT-PCR
Hemi-Nested
M13
ReNested MID
M13f M13r
13N5Bo8254
5Bo8254
13N5Bo8641
5Bo8707
C E1 E2 p7 NS2 NS3 NS5A NS5BNS4B4A
M13fOligo A+
TCAG
MID 5Bo8254 M13f TCAG+Oligo
B MID5Bo8641
Sanger High-resolution HCV subtyping (454/GS-Junior)
454nts
428nts
M13f
MIDOligo A+
TCAG
M13r
MID
TCAG+Oligo
B
45 490
M13f
M13UTR146
UTR45 Cor490
M13Core490
M13r
385nts
5’UTR-core
446nts
EU PATENT No. WO2015001068 A1
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Length:5’ core : 301ntsNS5B : 335 nts
HCV genotypes and subtypes. Region Discriminating power.
HCVIf genetic distance is lower than 10% subtype will not change
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HIGH-RESOLUTION HCV SUBTYPING
Original fasta file (GS-Junior software) RAW DATA includes60.000-192.000 sequences=reads (Passed filter wells)
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HIGH-RESOLUTION HCV SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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If genetic distance is lower than 10% subtype will not change
HIGH-RESOLUTION HCV SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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G1b
HIGH-RESOLUTION HCV SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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HIGH-RESOLUTION HCV SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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HIGH-RESOLUTION HCV SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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G4d
HIGH-RESOLUTION HCV SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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HIGH-RESOLUTION HCV SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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G1a
HIGH-RESOLUTION HCV SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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5
Patient P-IND-32 is infected by:
G4d (87%) + G1a (13%)
MIXED INFECTION = Infection by most than one subtype at the same time
HIGH-RESOLUTION HCV SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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SPECIAL PATIENT:
PATIENT INFECTED by 1b + 3a + 2c + 4d + 1a.
HIGH-RESOLUTION HCV SUBTYPINGCongreso Nacional del Laboratorio Clínico 2016
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HCV genome
3‘NCR5‘NCR
STRUCTURAL NO STRUCTURAL
NS3 (HCV RNA protease-helicase)
7602 9377
RT-PCR
Nested M13
ReNested MID (15 cycles)
M13f M13r
M13f M13r
MID MID
C E1 E2 p7 NS2 NS3 NS5A NS5BNS4B4A
M13fOligo A+ TCAG MID Up M13f TCAG+Oligo B MIDDown
Massive Sequencing 454/GS-Junior platform
Oligo A+ TCAG TCAG+Oligo B
3426 4036
M13f M13r
Primer N up
Primer up Primer down
611ntsPrimer N down
NS5A (replication
control)
6258 7601
NS5B (HCV RNA Dependent RNA
Polymerase)
M13f M13r
1344 nts
Primer up Primer downPrimer up Primer down
Primer N up Primer N down
Primer N up Primer N down
1776nts
DETECTION OF RAS = Resistant-associated amino acid SubstitutionsSP
ECIF
IC M
OD
ULE
UN
IVER
SAL
MO
DU
LECongreso Nacional del Laboratorio Clínico 2016
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NEXT GENERATION SEQUENCING (NGS) PLATFORMS
MASSIVE PARALLEL SEQUENCING
Platforms 2016:
Illumina (Solexa) sequencing: 300-500bp
Roche 454 sequencing (Roche): 400-1000bp
Ion torrent:Proton/PGM sequencing (Thermo Fisher Scientific): ~200bp
SOLiD sequencing (Applied Biosystems): 50-60bp
Platforms 2017:SINGLE MOLECULE REAL-TIME SEQUENCING (SMRT). Avenio Z1 (SMRT-NNGS Roche): 20.000nt Avenio Z1
NEXT-NEXT GENERATION SEQUENCING (NNGS)
Congreso Nacional del Laboratorio Clínico 2016
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DE NOVO SEQUENCING:Long sequencing reads (20000nts) will facilitate whole-genome sequencing (easy to assembly andto align). In many cases, reference sequences are not good enough and the genomes need to beresequenced.
WHOLE-GENOME SEQUENCING (100%):Thousands of complete viral genomes (HCV, HBV, HIV,…) could be generated.Highly DNA repetitive fragments could resolved (GpC isles, GGGGCC, ...)Large Insertions and/or Deletions could be resolved
WHOLE TRANSCRIPTOMA:All different mRNA isoforms transcribed from a single gene could be identified.
EPIGENETIC MODIFICATIONSEpigenetic modifications can be identified during DNA sequencing process without any previousDNA manipulation.
LONG SEQUENCING FRAGMENTS. A new sequencing paradigm?:
Congreso Nacional del Laboratorio Clínico 2016
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HCV genome
3‘NCR5‘NCR
STRUCTURAL NON STRUCTURAL
NS3 (HCV RNA protease-helicase)
9377
RT-PCR
Nested M13
ReNested MID (15 cycles)
M13rMIDM13f
MID
C E1 E2 p7 NS2 NS3 NS5A NS5BNS4B4A
M13fMID Up M13f MIDDown
3426
Primer up Primer down
NS5B (HCV RNA Dependent RNA
Polymerase)
M13f M13r
Primer NS up
Primer N S down
Ligation
M13fMID Up M13f MIDDown
SPEC
IFIC
MO
DU
LE
UN
IVER
SAL
MO
DU
LE
Congreso Nacional del Laboratorio Clínico 2016
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NEXT GENERATION SEQUENCING (NGS) PLATFORMS
MASSIVE PARALLEL SEQUENCING
Platforms 2016:
Illumina (Solexa) sequencing: 300-500bp
Roche 454 sequencing (Roche): 400-1000bp
Ion torrent:Proton/PGM sequencing (Thermo Fisher Scientific): ~200bp
SOLiD sequencing (Applied Biosystems): 50-60bp
Platforms 2017:SINGLE MOLECULE REAL-TIME SEQUENCING (SMRT). Avenio Z1 (SMRT-NNGS Roche): 20.000nt Avenio Z1
NEXT-NEXT GENERATION SEQUENCING (NNGS)
Platforms 201?:NANOPORE TECHNOLOGY
Congreso Nacional del Laboratorio Clínico 2016
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50
PEG-labelednucleotides
http://www.geniachip.com/
5 runs/day/plate$100/ genome
+ Columbia & Harvard University
Measure of current change caused by passage through the nanopore of each of the fourdifferent tags released during polymerase reaction = It generates an electronicsignature. To handle “read lengths” of several thousand nucleotide bases.
Alfa hemolisina (a-HL): bacterial toxin capable of forming pores in red blood cells and other cells causing cell lysis.
a-HL pore embedded in a lipid bilayer membrane
Congreso Nacional del Laboratorio Clínico 2016
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ONLY THE INCOMPETENT COMPETE,
THE COMPETENT COLLABORATEEudald Carbonell (Antropologist. Atapuerca’s Director)
Congreso Nacional del Laboratorio Clínico 2016
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Many thanks!LIVER DISEASE UNIT
VALL D’HEBRONINSTITUT OF
RESEARCH (VHIR)
HOSPITAL UNIVERSITARI VALL D’HEBRON (HUVH)
BARCELONA
Congreso Nacional del Laboratorio Clínico 2016
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WORKFLOW PRINCIPLES AVENIO Z1Congreso Nacional del Laboratorio Clínico 2016
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ZMW (Zero-mode-waveguide)
DNA pol+ template
up to 3 bases/sc )(
(20 zeptoliters (10-21 L)
P-nucleotides + fluorophore
Single-photon sensitive CCD array
recording in real time (30-240 min.
movies)
(1000000 ZMW/SMRT cell read in
parallel)
SMRT cell
≈20-30nm
Ø=70 nm
~100nm
SMRT-NNGSAVENIO Z1
Congreso Nacional del Laboratorio Clínico 2016
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Single Molecule Real-Time (SMRT) Sequencing.
https://www.youtube.com/watch?v=PQlodm9VwnI&authuser=0
Congreso Nacional del Laboratorio Clínico 2016
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VHIR-HUVH
SINGLE MOLECULE REAL-TIME - NEXT NEXT GENERATION SEQUENCING (SMRT-NNGS)
AVENIO Z1
Secuenciación de fragmentos de ADN de hasta : 500nucleótidos de
longitud
Una SMRT cell = 150.000 secuencias que equivale a 2 equipos 454/GS-Junior
SMRT-cell
Capacidad AVENIO Z1= 16 SMRT cellsEsto equivale a 32 equipos 454/GS-Junior
Secuenciación de fragmentos de ADN de hasta 20.000nucleótidos
RECAMBIO TECNOLÓGICO (Diciembre 2016)
454/GS-Junior
Congreso Nacional del Laboratorio Clínico 2016