development of a platform for hiv/hcv genotyping to anti-viral drug resistance using next generation...
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Development of a platform for HIV/HCV genotyping to anti-viral drug resistance using Next Generation Sequencing - NGS technology
Rodrigo de Moraes Brindeiro – UFRJAmilcar Tanuri – UFRJ
Mônica B. Arruda – UFRJ
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Fazer Genotipagem do HIV usando sequenciamento de nova geração (Next Generation Sequencing – NGS) vai me deixar na moda? Mesmo achando que é caro e complexo?
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Fazer Genotipagem do HIV usando sequenciamento de nova geração (Next Generation Sequencing – NGS) vai me deixar na moda? Mesmo achando que é caro e complexo?
Como usar NGS para fazer uma genotipagem completa do HIV – 5 alvos;Multiplex e de baixo custo!(e ainda agregar o HCV...)
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Rationale(1)• New ARV drugs against HIV, in new therapeutical classes, demand new
genetic targets for HIV genotyping:– Integrase: Raltegravir, Elvitegravir, Dolutegravir...– env gp41: Fuzeon® T20– env C2V3: Maraviroc, ...
• Comercial (FDA approved) Genotyping assays/kits using Sanger’s modified sequencing method do not evaluate all these genetic regions (targets) where DRMs accumulate:– Just Protease (PR) and Reverse Transcriptase (RT) genes
• Drug resistance genotyping based on ion torrent deep sequencing can evaluate all genetic regions (targets) where DRMs accumulate, by low cost:– Protease (PR) e Reverse Transcriptase (RT) (RNAseH region included),
Integrase, env gp41, env C2V3 and Gag (co-evolution of PR cutting sites and late domain)
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Rationale(1)• New ARV drugs against HIV, in new therapeutical classes, demand new
genetic targets for HIV genotyping:– Integrase: Raltegravir, Elvitegravir, Dolutegravir...– env gp41: Fuzeon® T20– env C2V3: Maraviroc, ...
• Comercial (FDA approved) Genotyping assays/kits using Sanger’s modified sequencing method do not evaluate all these genetic regions (targets) where DRMs accumulate:– Just Protease (PR) and Reverse Transcriptase (RT) genes
• Drug resistance genotyping based on ion torrent deep sequencing can evaluate all genetic regions (targets) where DRMs accumulate, by low cost:– Protease (PR) e Reverse Transcriptase (RT) (RNAseH region included),
Integrase, env gp41, env C2V3 and Gag (co-evolution of PR cutting sites and late domain)
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Rationale(1)• New ARV drugs against HIV, in new therapeutical classes, demand new
genetic targets for HIV genotyping:– Integrase: Raltegravir, Elvitegravir, Dolutegravir...– env gp41: Fuzeon® T20– env C2V3: Maraviroc, ...
• Comercial (FDA approved) Genotyping assays/kits using Sanger’s modified sequencing method do not evaluate all these genetic regions (targets) where DRMs accumulate:– Just Protease (PR) and Reverse Transcriptase (RT) genes
• Drug resistance genotyping based on NGS sequencing can evaluate all genetic regions (targets) where DRMs accumulate, by low cost:– Protease (PR) e Reverse Transcriptase (RT) (RNAseH region included),
Integrase, env gp41, env C2V3
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Rationale (2)• DRM Genotyping: not clonal, synergy between mutations not evaluated:
– Syntheny between mutations multi-resistant virus or– Mutations in different subpopulations mixture of resistant and wild type
viruses.
• ARV-resistant subpopulations present under 10-20% of total are not considered but can further impact on the therapy efficacy.
• The concept of depth of coverage (nber. of times a given sequence is obtained) de sequências clonais, through ion torrent sequencing, allows the evaluation of mutation occurence in viral sub-populations and thus:– Their impact on the efficacy of ARV therapy used, as well the new rescue
regimen to be implemented...– Minor sub-populations carrying DRMs, circulating at the threshold level of the
wild-type (ARV sensitive) major population;– The cell tropism of viral sub-populations (M-tropic x T-tropic,
or CCR5 x CXCR4 tropic) when analyzing the env C2V3 region; for the correct implementation of Maraviroc therapeutics 2-5% sub-population detection sensitivity
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Rationale (2)• DRM Genotyping: not clonal, synergy between mutations not evaluated:
– Syntheny between mutations multi-resistant virus or– Mutations in different subpopulations mixture of resistant and wild type
viruses.
• ARV-resistant subpopulations present under 20% of total are not considered but can further impact on the therapy efficacy.
• The concept of depth of coverage (nber. of times a given sequence is obtained) de sequências clonais, through ion torrent sequencing, allows the evaluation of mutation occurence in viral sub-populations and thus:– Their impact on the efficacy of ARV therapy used, as well the new rescue
regimen to be implemented...– Minor sub-populations carrying DRMs, circulating at the threshold level of the
wild-type (ARV sensitive) major population;– The cell tropism of viral sub-populations (M-tropic x T-tropic,
or CCR5 x CXCR4 tropic) when analyzing the env C2V3 region; for the correct implementation of Maraviroc therapeutics 2-5% sub-population detection sensitivity
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SANGER Sequencing technologies:
• Not clonal: blend of populations.
• Do not discriminate subpopulations
• Not sensitive to detect mutations in minor subpopulations (under 20%)
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Rationale (2)• DRM Genotyping: not clonal, synergy between mutations not evaluated:
– Syntheny between mutations multi-resistant virus or– Mutations in different subpopulations mixture of resistant and wild type
viruses.
• ARV-resistant subpopulations present under 20% of total are not considered but can further impact on the therapy efficacy.
• The concept of depth of coverage (nber. of times a given sequence is obtained) of clonal sequences, through NGS sequencing, allows the evaluation of mutation occurence in viral sub-populations and thus:– Their impact on the efficacy of ARV therapy used, as well the new rescue
regimen to be implemented...– Minor sub-populations carrying DRMs, circulating at the threshold level of the
wild-type (ARV sensitive) major population;– The cell tropism of viral sub-populations (M-tropic x T-tropic,
or CCR5 x CXCR4 tropic) when analyzing the env C2V3 region; for the correct implementation of Maraviroc therapeutics 5-10% sub-population detection sensitivity
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Rationale(3)• OTHERS:
– Compatible cost.. aggregate HCV (hepatitis C) Protease and Replicase; to evaluate:
• viral genotype impact on therapy• DRMs for replicase and protease inhibitors (Ribavirine,
Boceprevir, Telaprevir, ...)
Um único Kit, vários alvos,
dois (ou mais?) vírus...
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Brief• Today:
– Genotyping of HIV by comercial methods (Sanger):
• Two (only) therapeutic targets• Cost to MoH: aprox. U$ 125,00 to U$ 150,00 /
genotyping
– Genotyping HCV:• No assay
(in house methods, in some cases)
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Brief
• Perspective NGS -Deep Sequencing: – Genotyping HIV using NGS:
• ALL therapeutic targets (actual: 5-6)• Cost to MoH:
– aprox. 1 chip (10-100Mb) multiplexed (12-20 patients/barcodes; coverage of 250x-1000x)
– U$ 1.000,00 to U$ 1.500,00 / rxn = U$ 70,00 to U$ 100,00 / patient.
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Brief• Perspective Deep Sequencing:
– Genotyping HCV using NGS:
• ALL therapeutic targets (actual: 2)• Cost to MoH:
– aprox. same chip (100Mb) multiplexed (12 patients/barcodes; coverage of 250-1000x); OR
– other chip? HBV included? TB?
- Aprox. 70.000 cases until 2010– Aprox. 30.000 in treatment– Aprox. 40% (12.000) under treatment failure
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R&D
• deep sequencing test for resistance genotyping in HIV:
– R&D: Solexa illumina MiSeq –comercial kit– Roche: 454 kit, HIV PR & RT only (??)– Basic Science (inhouse) R&D – Spain
(plataform 454) – Roger Paredes, PhD (group of Bonaventura Clotet, PhD MD, chief of University
Hospital Germans Trias i Pujol in Barcelona, Spain)
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Pirossequenciamento Roche 454 – GS Junior FLX
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Pirossequenciamento
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Pirossequenciamento
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Illumina MiSeq / HiSeq
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Illumina
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Illumina
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Ion Torrent - ionograma
• Platform:
• Ion PGMTM < Ion ProtonTM
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Ion Torrent
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Comparação de tecnologias
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• Chips:314
316
318
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• Chips:314
316
318
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• 314:
– milhões de sensores– 20 Mb read/coverage– Amplicons 200bp (400bp ideal next?)
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Inicial...• 316:
– 7 milhões de sensores
– 200 Mb de cobertura total
– Amplicons de 200pb (400pb ideal possível?)
– Amplicons HIV: 17 (3 GAG, 2 PR, 6 RT, 3 IN, 2 ENVgp41, 1 ENVgp120) ou,
11 (2 GAG, 1 PR, 4 RT, 2 IN, 1 ENVgp41, 1
ENVgp120)
– Amplicons HCV: 10 (4 PR, 6 Rep) ou,
6 (2 PR, 4 Rep)
– Total: 27 ou 17
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• 316:
– Total: 27 ou 17 amplicons– BIDIRECIONAL (A + B-P1 adaptors para ambos primers F e R)– Capacidade: 250 amplicons com cobertura 1000-2000x, logo– Capacidade de detectar até 5% de variação (cut-off aceitável) – 20 a 40
leituras de 5% por região.– 27amps x 200(165)pb x 1000 cob. = 5.4Mb ou– 17amps x 400(365)pb x 1000 cob = 6.8Mb– 5.4Mb x 18 genos/pacientes = 97.2Mb ou– 6.8Mb x 14 genos/pacientes = 95.2Mb
Ion DNA Barcoding 1-16 Kit (10 sets of 16 libraries)
17-n Kit
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• 316:
– Total: 27 ou 17 amplicons– bottleneck:
» Ancoramento dos diversos primers (54 ou 34!!!) em regiões genômicas estáveis (TODAS!)...
» Alguma saída?
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• 316:
– Total: 27 ou 17 amplicons– bottleneck:
» Ancoramento dos diversos primers (54 ou 34!!!) em regiões genômicas estáveis (TODAS!)...
» Alguma saída?
CONTINGÊNCIA: o PLANO B!
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• 314:
– METHOD: AMPLICON X LIBRARY» Preparation of 2 subgenomic amplicons of HIV (~4kb: 3kb PR-RT-
INT, 1kb ENV); enzyme break and library prep (adaptor ligation) » HCV: 1 amplicon (3kb PR-Replicase); enzyme break and library
prep (adaptor ligation) » AB Library Builder™ System
– Ion Xpress™ Fragment Library Kit (up to 20 reactions)– Ion One Touch System– PGM System
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Library Builder & Ion One Touch
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Long Amplicons: SuperScript III + High Fidelity Platinum Taq one step
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• 314:
– Costs:
» Chip: aprox. U$ 800,00
» All rxns: U$ 300,00 to U$ 500,00
» Consumables: polymerases, RT-PCR kits, plasticware, dNTP, etc.
» Genotyping/patient: ~U$ 1.200/12 genos=
aprox. U$ 100,00
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Software• Initial (raw data mining):
• CLC ®
• Geneious ®
• Proprietary ion torrent ®
• Other? (interpretation algorythm)
• Genotyping Reports:– New Development:
» Language: VbNET? Delphi? Java?
» Data bank: MySQL? SQL Server?
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Calendar of Activities
Ano 2012 2013Mês Mai Jun Jul Ago Set Out Nov Dez Jan Fev Mar AbrMês # 1 2 3 4 5 6 7 8 9 10 11 12
Atividades(a) Desenho de primers
e kit rationale(b) Instalação e treino
IonTorrent(c ) Testes PCR
sensibilidadeespecificidade(subtipos, variantes)
(d) Testes ion torrentsensibilidadeespecificidademisturas popul.
(e) Desenvolvimentode software
(f) piloto em sítiolaboratorial
(g) Multicêntrico em labs de Rede
Submissões de projeto
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Project Costs• Plataforma Ion Torrent• Kits / Chips 316;314:
– Fase I: PROVA de CONCEITO• treinamento e 1os testes e erros: 06 chips/kits• Prova de conceito (multiplex, barcode): 04 chips/kits• Testes de subtipos/genótipos/variantes: 04 chips/kits• Sensibilidade (LOD): 10 chips/kits• Sensibilidade de detecção misturas (subpopulações): 04 chips/kits
– Fase II: ESTUDOS CLÍNICOS de VIABILIDADE• Estudo Piloto (sítio de rede labs) 04 chips/kits • Estudo Multicêntrico (3 sítios de rede) 16 chips/kits
– Mais: material de suporte (plasticaria, kits p/PCR)...– FASE II: submissão de projeto PPP: FINEP, BNDES, FAPERJ
(também na FASE I final? Prova de conceito estabelecida?)– Bolsas (2) de pesquisador
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PerspectivesNew Joint Projects:
1. VL assay for HIV/HCVusing digital PCR
2. Malignant Hyperthermia (Anesthesics in surgery problem) diagnostic using SNPs on digital PCR
Bottlenecks:Competition - miniaturization- point-of-care:- µfluidics +- enzymes estabilization in chip =- lab-on-chip- easy-to-use “credit card handle machines” and “iPhone” devices for mol.biology!!! Lab-on-foils, lab-on-chips.
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