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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 – UFRJ
Amilcar Tanuri – UFRJ
Mônica B. Arruda – UFRJ
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?
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...)
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)
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)
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
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
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
SANGER Sequencing
technologies:
• Not clonal: blend of populations.
• Do not discriminate
subpopulations
• Not sensitive to detect mutations
in minor subpopulations (under
20%)
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
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...
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)
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.
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
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)
Pirossequenciamento
Roche 454 – GS Junior FLX
Pirossequenciamento
Pirossequenciamento
Illumina MiSeq / HiSeq
Illumina
Illumina
Ion Torrent - ionograma
• Platform:
• Ion PGMTM < Ion ProtonTM
Ion Torrent
Comparação de tecnologias
• Chips:
314
316
318
• Chips:
314
316
318
• 314:
– milhões de sensores
– 20 Mb read/coverage
– Amplicons 200bp (400bp ideal next?)
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
• 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
• 316:
– Total:
27 ou 17 amplicons
– bottleneck:
» Ancoramento dos diversos primers (54 ou 34!!!) em regiões
genômicas estáveis (TODAS!)...
» Alguma saída?
• 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!
• 314:
– METHOD: AMPLICON X LIBRARY
» Preparation of 2 subgenomic amplicons of HIV (~4kb: 3kb PRRT- 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
Library Builder & Ion One Touch
Long Amplicons:
SuperScript III + High Fidelity Platinum Taq one step
• 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
Software
• Initial (raw data mining):
• CLC ®
• Geneious ®
• Proprietary ion torrent ®
• Other? (interpretation algorythm)
• Genotyping Reports:
– New Development:
» Language:
» Data bank:
VbNET? Delphi? Java?
MySQL? SQL Server?
Calendar of Activities
Ano
Mês
Mês #
2012
Mai
1
Jun
2
Jul
3
Ago
4
Set
5
Out
6
Nov
7
Atividades
(a)
Desenho de primers
e kit rationale
(b)
Instalação e treino
IonTorrent
(c )
Testes PCR
sensibilidade
especificidade
(subtipos, variantes)
Testes ion torrent
(d)
sensibilidade
especificidade
misturas popul.
(e)
Desenvolvimento
de software
(f)
piloto em sítio
laboratorial
(g)
Multicêntrico em
labs de Rede
Submissões de projeto
Dez
8
2013
Jan
9
Fev
10
Mar
11
Abr
12
Project Costs
• Plataforma Ion Torrent
• Kits / Chips 316;314:
– Fase I: PROVA de CONCEITO
•
•
•
•
•
treinamento e 1os testes e erros:
Prova de conceito (multiplex, barcode):
Testes de subtipos/genótipos/variantes:
Sensibilidade (LOD):
Sensibilidade de detecção misturas (subpopulações):
06 chips/kits
04 chips/kits
04 chips/kits
10 chips/kits
04 chips/kits
– Fase II: ESTUDOS CLÍNICOS de VIABILIDADE
• Estudo Piloto (sítio de rede labs)
• Estudo Multicêntrico (3 sítios de rede)
04 chips/kits
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
Perspectives
New Joint Projects:
1. VL assay for HIV/HCV
using 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.