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Secuenciación masiva en el estudio de las enfermedades hepáticas: aplicaciones y retos Josep Quer [email protected] Liver Unit. Vall d’Hebron Institut of Research (VHIR). Hospital Universitari Vall d’Hebron (HUVH). Univers. A utònoma de Barcelona. Ciber Enfermedades Hepáticas y Digestivas (Ciberehd) del Instituto de Salud Carlos III. Madrid. Sociedad Española de Virologia (SEV). MASSIVE SEQUENCING? MASSIVE SEQUENCING or NEXT GENERATION SEQUENCING (NGS) or DEEP SEQUENCING or MASSIVE 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). Platforms 2015 (size of reads): üIllumina (Solexa) sequencing: 100-‐150bp üRoche 454 sequencing (Roche): 400-‐1000bp üIon torrent:Proton/PGM sequencing (Thermo Fisher Scientific): ~200bp üSOLiD sequencing (Applied Biosystems): 50-‐60bp History of Human Genome Sequencing. From SANGER to NGS Human Genome Project (HGP). Public Consortium 13 years (3Gb) 1950 1960 1953 discovery Double helix structure of DNA Rosalind Elsie Franklin, Watson y Crick 1970 1980 1977 1983 Sanger PCR sequencing Kary Mullis method 1990 1990 HGP Start 2015: One human genome (3Gb=3000Mb) can be sequenced within a single day James Watson´s genome 1 month (3Gb) 2000 2003 HGP Completed Classical Sanger Sequencing 2007 NGS 5 days (5Gb= 1,5 genomes) 2009 NGS 1 human genome = 3000Mb = 3 Gb ü2504 individuals üFrom 26 populations ü84.7M SNPs (single nucelotide polymorphisms) ü3.6 M INDELS (insertions and deletions) ü60,000 structural variants 1000 Genomes Project Consortium, A uton A, et al. Nature 2015;526;7571;68-‐74 EXAMPLE OF CLINICAL APPLICATION of MASSIVE PARALLEL SEQUENCING Gustavo Palacios et al NEJM 2008;;358(10):991-8 To sequence a DNA or RNA genome we only need to know both ends. Single stranded library 500000 cells NEJM 2008;;358(10):991-8 Se identificó un nuevo ARENAVIRUS relacionado con el Virus de la Coriomeningitis Linfocítica. SOME BIG SEQUENCING PROJECTS http://www.1000geno mes.org Secuenciación masiva en el estudio de las enfermedades hepáticas: aplicaciones y retos CAUSES OF LIVER DISEASE (>30) Algille síndrome (genetic alteration) Alcohol hepatopathy (drugs or toxins) Alpha 1 Anti-‐Trypsin Deficiency (genetic alteration) Amebic liver abscess (parasite Entamoeba histolytica) (infectious agent) Autoimmune Hepatitis ( not infectious, generally not considered an inherited, ? ??) Biliar Atresia (complex process, not genetic alteration) Cirrhosis (multiple causes, infectious agents, genetics, drugs, toxins) Coccidioidomycosis or Valley fever (spores fungus Coccidioides immitis) (infectious agent) Cystic disease of t he liver: Caroli’s syndrome (inherited) ; Congenital hepatic fibrosis (probably inherited) Drug-‐induced liver injury (drugs or toxins) Drug-‐induced cholestasis (drugs or toxins) Fattly liver disease (Disease progression depends of a combination of genetic and environmental factors and Alcoholism Viral infections (HBV, HCV, HDV) Metabolic diseases: (alpha-‐1 -‐antitrypsin deficiency, galactosemia and glycogen storage disorders) Wilson disease Hemochromatosis, Primary biliary cirrhosis (PBC) Primary sclerosing cholangitis PSC) Toxic hepatitis (drugs, toxins and repeated bouts of heart failure with l iver congestion.) is a ffected b y l ifestyle c hoices a nd o ther factors such a s the gut microbiome . Galactosemia (genetic alteration) Gallstones (unknown, genetic alteration?) Gilbert´s Syndrome (genetic alteration) Hemochromatosis (genetic alteration) Liver CANCER: hepatocellular carcinoma (HCC), cholangiocarcinoma, metastases (multiple c auses, infectious a gents, g enetics, epigenetic changes, d rugs, toxins…). Liver disease in pregnancy (unknown) Lysosomal Acid Lipase Deficiency (LALD) (genetic alteration) Neonatal Hepatitis (virus?) Primary Biliary Cirrhosis (autoimmune caused by a virus?) Primary Sclerosing Cholangitis (genetic alteration) Porphyria (genetic alteration) Pyogenic liver abscess (infectious agent) Reye´s Syndrome (infectious agent, genetic alteration?) Sarcoidosis (unknown cause) Toxic Hepatitis (chemicals =drugs, industrial solvents and pollutants) Type 1 Glycogen Storage Disease (genetic alteration) Tyrosinemia (genetic alteration) Wilson Disease (genetic alteration) VIRAL HEPATITIS (infectious agent) Molecular “SIGNATURE”: -‐Patient stratification -‐Prognosis value -‐Treatment selection -‐Biomarker identification (a m esurable indicator of t he severity or presence of some disease state) Rodés J & Guardia J M ed.Interna Tomos I y II EASL.HEPAMAP: Martin P. Approach to the patient with liver disease. In: Goldman L, S chafer AI, eds. Goldman's Cecil M edicine. 24th ed. Philadelphia, PA: Saunders Elsevier; 2011:chap 148. https://www.nlm.nih.gov http://www.liver.ca/liver-‐disease/types/ https://www.nlm.nih.gov/medlineplus/ency/article/000205.htm E N F E R M E D A D GENOMA H E P Á T I C A FENOTIPO Ritchie MD et al Nat Reviews/Genetics 2015; 1 6:85-‐97 MOLECULAR SIGNATURES for HCC: -Patient stratification -Prognosis value -Treatment selection GENOMA E N F E R M E D A D H E P Á T I C A FENOTIPO Ritchie MD et al Nat Reviews/Genetics 2015; 1 6:85-‐97 GENOMA E N F E R M E D A D H E P Á T I C A FENOTIPO Ritchie MD et al Nat Reviews/Genetics 2015; 1 6:85-‐97 Methylation along HBV integrated viral genome. From chronic infection to advanced HCC. Genome Research 2009;; 19:438-451 GENOMA E N F E R M E D A D H E P Á T I C A FENOTIPO Ritchie MD et al Nat Reviews/Genetics 2015; 1 6:85-‐97 GENOMA E N F E R M E D A D H E P Á T I C A FENOTIPO Ritchie MD et al Nat Reviews/Genetics 2015; 1 6:85-‐97 MOLECULAR SIGNATURES in Liver Disease: - long non-coding RNAs - Micro RNA ENVIRONMENT FROM GENOME TO PHENOTYPE PRECISION MEDICINE E N F E R M E D A D GENOMA H E P Á T I C A FENOTIPO MASSIVE PARALLEL SEQUENCING Ritchie MD et al Nat Reviews/Genetics 2015; 16:85-‐97 Big data: Storage, Methods of Integrating data, Data managing (BIOINFORMATICS) Translational Biomedical Research WORKFLOW: From patient to final Report RNA/DNA Isolation Capp Med Target Enrichment 2nd and 3rd Analysis GeneWEAVE Library Preparation Sequencing. New platforms Reporting Roche-‐ AVENIO Translational Biomedical Research WORKFLOW: From patient to final Report RNA/DNA Isolation 1.-‐ EXPERIMENT DESIGN (selection of the best targets) 2nd and 3rd Analysis Target Enrichment 2.-‐ SAMPLING: “the less invasive the best“ Library Preparation Sequencing. New platforms Reporting WORKFLOW: FROM GENOME TO PHENOTYPE RNA/DNA Isolation Capp Med Target Enrichment 2nd and 3rd Analysis GeneWEAVE Library Preparation Sequencing. New platforms Reporting Roche-‐ AVENIO La investigación Biomédica Traslacional requiere del trabajo coordinado de equipos MULTIDISCIPLINARIOS formados por profesionales de diferentes ámbitos. CAUSES OF LIVER DISEASE HEPAMAP: EASL http://www.easl.eu/medias/E ASLimg/News/EASL_HEPAMA P_Full_Report.pdf Algille síndrome (genetic alteration) Alcohol hepatopathy (drugs or toxins) Alpha 1 Anti-‐Trypsin Deficiency (genetic alteration) Amebic liver abscess (parasite Entamoeba histolytica) (infectious agent) Autoimmune Hepatitis (not infectious, generally not considered an inherited, genetic alteration) Biliar Atresia (complex process, not genetic alteration) Cirrhosis (multiple causes, infectious agents, genetics, drugs, toxins) Coccidioidomycosis or Valley fever (spores fungus Coccidioides immitis) (infectious agent) Cystic disease of t he liver: Caroli’s syndrome (inherited) ; Congenital hepatic fibrosis (probably inherited) Drug-‐induced liver injury (drugs or toxins) Drug-‐induced cholestasis (drugs or toxins) Fattly liver disease (Disease progression depends of a c ombination of genetic and environmental factors and i s affected by l ifestyle c hoices and other factors such a s the gut microbiome . Galactosemia (genetic alteration) Gallstones (unknown, genetic alteration?) Gilbert´s Syndrome (genetic alteration) Hemochromatosis (genetic alteration) Liver CANCER: hepatocellular carcinoma (HCC), cholangiocarcinoma, metastases (multiple causes, infectious agents, genetics, epigenetic c hanges, drugs, toxins…). Liver disease in pregnancy (unknown) Lysosomal Acid Lipase Deficiency (LALD) (genetic alteration) Neonatal Hepatitis (virus?) Primary Biliary Cirrhosis (autoimmune caused by a virus?) Primary Sclerosing Cholangitis (genetic alteration) Porphyria (genetic alteration) Pyogenic liver abscess (infectious agent) Reye´s Syndrome (infectious agent, genetic alteration?) Sarcoidosis (unknown cause) Toxic Hepatitis (chemicals =drugs, i ndustrial solvents and pollutants) Type 1 Glycogen Storage Disease (genetic alteration) Tyrosinemia (genetic alteration) Wilson Disease (genetic alteration) VIRAL HEPATITIS (infectious agent) Genetic “SIGNATURE”: -‐Genetic alteration -‐Epigenetics -‐Genetic predisposition -‐Effect of gut microbiota (Llopis M et al Gut 2015; Dec 15. I ntestinal microbiota contributes to individual susceptibility to alcoholic liver disease) ... -‐Infectious agents: VIRUS... THE MINIMUM DISTANCE BETWEEN GENOME AND PHENOTYPE= VIRUS Example HEPATITIS C Virus (HCV) 93 HCV plasma pre-‐treatment with NS5Ai HCV plasma at failure after treatment with NS5Ai QUASISPECIES. POPULATION OF SEQUENCES MASSIVE PARALLEL SEQUENCING = thousand of seqs BEST TOOL to STUDY population of sequences SANGER SEQUENCING = 1 seq (CONSENSUS) 1 to 1010-1012 CONSENSUS 1 24 COMBINATION THERAPY IS THE BEST STRATEGY TO FIGHT AGAINST HIGHLY VARIABLE SYSTEMS: -RNA VIRUS - ONCOLOGY RNA VIRUS ONCOLOGY 1989 Esteban Domingo J.Hepatol 2012; 56:1343-‐50 http:www.hepmag.com HCV DRUG TARGETS. DAA = Direct Acting Antivirals STRUCTURAL NO STRUCTURAL 3‘UTR 5‘NCR PROTEASE Inhibitors (PI) Block active site viral enzyme -‐PREVIR LOW BR 1st Gen.1st wave (G1): TELAPREVIR (TVR) * BOCEPREVIR (BOC) * 1st Gen.2nd wave (all G except 3) SIMEPREVIR (SMV) * ASUNAPREVIR & PARITAPREVIR/ritonavir* VANIPREVIR (MK-‐7009) DANOPREVIR HIGHER BR 2nd Generation. Pangenotyping less efective for G3: MK-‐5 172 (Grazoprevir-‐GRZ) # ACH-‐2 684 NS5A Inhibitors (NS5AI) Block Replication complex, particle assembly & release -‐A SVIR LOW BR 1st Gen. (G1 and G4. Other genotypes variable) DACLATASVIR (DCV) * LEDIPASVIR * OMBITASVIR (ABT-‐2 67) * Slighlty HIGHER BR 2nd Gen. (Pangenotyping) MK-‐8 742 (Elbasvir-‐EBR) # GS-‐5 816 (Velpatasvir-‐VEL) # ACH3102 SAMATASVIR (IDX719) October 10th, 2014 F DA / December 15th 2014 AEMPS ORAL I FN-‐free treatment. HARVONI: LEDIPASVIR+SOFOSBUVIR(1pill). NS5B Non-‐Nucloes(t)ide Inhibitors (Non-‐ Nucs or NNI) Allosteric site. Change tridimensional structure -‐BUVIR NS5B Nucloes(t)ide Inhibitors (Nucs or NI) Block active site. Inhibits RNA elongation -‐BUVIR HIGH BR Pangenotyping (less SVR G3). GS-‐7 977=SOFOSBUVIR (SOF) * MERACITABINE # ACH-‐3 422 IDX21437 IDX21459 LOW BR Narrow genotyping coverage DASABUVIR (ABT-‐3 33)* DELEOBUVIR # BMS-‐7 91325 (Beclabuvir) # PPI-‐3 83 GS-‐9 669 TMC647055 December 19th 2014 FDA / M arch 2015 AEMPS ORAL I FN-‐free treatment (+/-‐Ribavirina). VIEKIRAX: OMBITASVIR-‐PARITAPREVIR-‐ritonavir (2p/1xday) + EXVIERA: DASABUVIR (1pill twice a day) In real life: 5-‐10% of TREATMENTS are FAILING AT FAILURE, RESISTANCE MUTATIONS (RAVs) are selected, and can be CROSS-‐RESISTANT to other INHIBITORS of the same family!!! 26 ... CUSTOMIZED TREATMENTS. NEED OBJECTIVE DATA PRECISION MEDICINE vVIRUS vPATIENT vCOMBINATION OF INHIBITORS (DAAs) VIRUS: ØSUBTYPE ØMIXED INFECTIONS ØRESISTANCE MUTATIONS MASSIVE PARALLEL SEQUENCING Sarrazin C. J.Hepatol. 2016;;64:486-504 PATIENT: ØFIBROSIS DEGREE ØTREATMENT-‐EXPERIENCED ØDRUG INTERACTIONS 27 CDTI (Centro para el Desarrollo Tecnológico Industrial), Spanish Ministry of Economics and Competitiveness (MINECO) CDTI Project Ref. IDI-‐20110115 IP. JUAN IGNACIO ESTEBAN HCV and HBV quasiespecies and genetics polymorphism studies associated to response to antiviral treatment using Pyrosequencing. ALLOWS: HCV Subtyping (to identify 67 subtypes) and Detection of Mixed Infections (more than one HCV subtype in a serum sample) 454 / GS-Junior v60000-192.000 sequences=reads (Passed filter wells) v 500-800nts Quer J et al. J.Clin.Microbiol. 2015; Jan 53(1):219-‐26 Antonio Madejón Javier García-‐Samaniego Ricardo Moreno Paloma Sanz-‐Cameno Rosa López-‐Rodríguez Sofía Pérez del Pulgar Xavier Forns Manolo Romero-‐Gómez Jose Antonio del Campo Esteban Domingo Celia Perales Julie Sheldon Francisco Rodríguez-‐Frías María Buti David García-‐Cehic Rosario Casillas María Blasi María Homs David Tabernero Leo Nieto María Cubero Silvia Sauleda Marta Bes Andrea Caballero Rafael Esteban Jaume Guardia Juan Ignacio Esteban Carlos Briones Jordi Gómez Javier Salmerón Paloma Muñoz de Rueda Rosa Quiles-‐Pérez Ángela Extremera Chalom Sayada Dimitri González Josep Gregori Miguel Alvárez-‐Tejado José Manuel Muñoz CENTRAL LABORATORIES Dr. Ernesto Casis MICROBIOLOGY DEPARTMENT Dr. Tomas Pumarola TRASLATION LIVER PATHOLOGY UNIT Dr. Francisco Rodríguez Frías Left-‐right: Maria Buti, Josep Gregori, Damir Garcia-‐Cehic, Francisco Rodríguez-‐Frias, Joan Ignasi Esteban, Josep Quer and Rafael Esteban 30 SUMMARY: USE OF MASSIVE PARALLEL SEQUENCING TO STUDY HCV INFECTION Ø HCV Subtyping identification of 67 subtypes. Re-‐classification can be performed if references are updated Ø Detection of Mixed Infections. Ø PROFILE OF RESISTANCE MUTATIONS. Ø To perform EPIDEMIOLOGICAL studies. 31 Secuenciación masiva. 2016 NEXT-‐NEXT GENERATION SEQUENCING SINGLE MOLECULE REAL-‐TIME (SMRT) sequencing Roche-‐AVENIO MASSIVE PARALLEL SEQUENCING. 2016 Roche-‐AVENIO One SMRT cell = 400,000 to 1,000,000 reads (5-‐12 GS-‐Junior) Roche-‐AVENIO DNA fragments 100bp-‐20kb Circular sequencing (max.70kb) 20,000nts x 1,000,000reads = 20,000,000,000nts = 6 human genomes/SMRTcell Roche-‐AVENIO = 16 SMRT cells Secuenciación De novo, genoma completo: Las lecturas largas (20,000nts) permitirán realizar la secuenciación de genomas completos, gracias a la facilidad de ensamblado y alineamiento. Soluciona la lectura de zonas altamente repetitivas (islas GC;; GGGGCC…) Roche-‐AVENIO NANOPORE TECHNOLOGY ¿ ? + Columbia & Harvard University http://www.geniachip.com/ PEG-‐labeled nucleotides a-‐HL pore embedded in a lipid bilayer membrane Measure of current change caused by passage through the nanopore of each of the four different tags released during polymerase reaction = It generates an electronic signature. 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. 5 runs/day/plate $100/ genome Many thanks! LIVER DISEASE UNIT VALL D’HEBRON INSTITUT OF RESEARCH (VHIR) HOSPITAL UNIVERSITARI VALL D’HEBRON (HUVH) BARCELONA ONLY THE INCOMPETENT COMPETE, THE COMPETENT COLLABORATE Eudald Carbonell (Catalan Antropologist. Atapuerca’s CoDirector) “Science is a choral activity, in which Multidisciplinary teams share the same lyrics/objectives”
