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

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 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

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: VbNET? Delphi? Java?

» Data bank: MySQL? SQL Server?

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

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

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.