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 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): Genotyping Reports: CLC ® Geneious ® Proprietary ion torrent ® Other? (interpretation algorythm) Genotyping Reports: New Development: Language: VbNET? Delphi? Java? Data bank: MySQL? SQL Server?

Calendar of Activities 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

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.

obrigado