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
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
Congreso Nacional del Laboratorio Clínico 2016
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
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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
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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
Congreso Nacional del Laboratorio Clínico 2016
19HCV
2.- COMPENSATORY MUTATIONS IN THE SAME GENOME CAN RETRIEVE LOST FITNESS
Congreso Nacional del Laboratorio Clínico 2016
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
EASL guidelines 2014EASL guidelines 2015
RAS detectionCongreso Nacional del Laboratorio Clínico 2016
2016 RAS detectionCongreso Nacional del Laboratorio Clínico 2016
2016 RAS detectionCongreso Nacional del Laboratorio Clínico 2016
RAS detection
2017
¿?
Congreso Nacional del Laboratorio Clínico 2016
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
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
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
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UN
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SAL
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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)
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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?:
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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
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
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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
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ONLY THE INCOMPETENT COMPETE,
THE COMPETENT COLLABORATEEudald Carbonell (Antropologist. Atapuerca’s Director)
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Many thanks!LIVER DISEASE UNIT
VALL D’HEBRONINSTITUT OF
RESEARCH (VHIR)
HOSPITAL UNIVERSITARI VALL D’HEBRON (HUVH)
BARCELONA
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WORKFLOW PRINCIPLES AVENIO Z1Congreso Nacional del Laboratorio Clínico 2016
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
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Single Molecule Real-Time (SMRT) Sequencing.
https://www.youtube.com/watch?v=PQlodm9VwnI&authuser=0
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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
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