Presentación de PowerPointlabclin2016.pacifico-meetings.com/images/site/Ponencias_LabClin2… · 17 HCV FITNESS MEASURES for different SUBSTITUTIONS. Studies in subgenomic replicon

Aplicaciones de la secuenciación masiva

en el manejo de la hepatitis C

Josep Quer

[email protected]

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


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)


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


http://en.wikipedia.org/wiki/Image:Sequencing.jpg

SOME BIG SEQUENCING PROJECTS

http://www.1000genomes.org

TCGA project

April 2016


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


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:


9

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


HCV is NOT a single SEQUENCE, but a POPULATION!

HCV


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


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


15HCV

DNA ARCHIVEResistance mutations may

be archived

No ARCHIVE.Persistance of a Resistance

mutant depends on itsFITNESS


16HCV

1.- FREQUENCY at which an HCV ANTIVIRAL RESISTANCE MUTANT is observed depends of its

FITNESS!!!


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




Insights from deep sequencing. RAS mutations


19HCV

2.- COMPENSATORY MUTATIONS IN THE SAME GENOME CAN RETRIEVE LOST FITNESS




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)

*


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!!!


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


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

¿?


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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If genetic distance is lower than 10% subtype will not change


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G1b


38


39


40

G4d


41


42

G1a


43

5

Patient P-IND-32 is infected by:

G4d (87%) + G1a (13%)

MIXED INFECTION = Infection by most than one subtype at the same time


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SPECIAL PATIENT:

PATIENT INFECTED by 1b + 3a + 2c + 4d + 1a.


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


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


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

NEXT GENERATION SEQUENCING (NGS) PLATFORMS


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)


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


M13fMID Up M13f MIDDown

3426

Primer up Primer down


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


NEXT GENERATION SEQUENCING (NGS) PLATFORMS


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


ONLY THE INCOMPETENT COMPETE,

THE COMPETENT COLLABORATEEudald Carbonell (Antropologist. Atapuerca’s Director)


Many thanks!LIVER DISEASE UNIT

VALL D’HEBRONINSTITUT OF

RESEARCH (VHIR)

HOSPITAL UNIVERSITARI VALL D’HEBRON (HUVH)

BARCELONA


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


https://www.youtube.com/watch?v=PQlodm9VwnI&authuser=0

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


Documents

Presentación de PowerPointlabclin2016.pacifico-meetings.com/images/site/Ponencias_LabClin2… · 17 HCV FITNESS MEASURES for different SUBSTITUTIONS. Studies in subgenomic replicon