Illumina Genome Analyzer. Progenika Experience. - Susana Catarino -

Transcription

1 Illumina Genome Analyzer Progenika Experience - Susana Catarino -

2 Who are we? 2000 PROGENIKA BIOPHARMA Development, production and commercialization of new genomic tools for diagnosis, prognosis and drug-response prediction of human diseases 2002 PROTEOMIKA Development of new proteomic tools for diagnosis, prognosis and drugresponse prediction of human diseases 2007 ABYNTEK Development, production and commercialization of recombinant proteins and monoclonal and polyclonal antibodies 2007 PROGENIKA Inc. Validates and commercializes PROGENIKA products in the US market 2008 BRAINco Develops new drugs for the treatment of psyquiatric disorders

3 Own Products - microarray for the diagnosis of Familial Hypercholesterolemia. It was released in Europe in June was developed to improve the safety of transfusions by genetically characterizing blood group antigens. - is a tool for predicting drug response in a broad array of metabolism pathways to improve safety and effectiveness of therapeutic strategies.

4 Human Resources Constant Growth 2004: 29 people 2005: 39 people 2006: 62 people 2007: 104 people 2008: 124 people 2009: 136 people Staff highly qualified 39% PhDs 10 Nationalities 9% MBA

5 Technological Resources Genomics (Progenika biopharma, SA. Progenika Inc.) Spotting DNAchips Affymetrix Luminex Illumina Applied Biosystems Tecan Bioinformatics

6 Funcional Genomics Services Expression Assays (Affymetrix) Genotyping (Illumina, Kaspar, TaqMan, Restriction Enzymes) Massive Sequencing (Illumina)

7 Genome Analyzer We use the technology since the end of During 2008 we used it for internal projects. We experienced a lot of problems in the begining. In 2009 we started to provide Massive Sequencing Services. Universities Research Centers Companies

8 vonwillebrand Project BST Barcelona - Progenika vonwillebrand disease (congenital coagulopathy) It takes its name from Dr. Erik von Willebrand, who first described the condition in vonwillebrand disease is the most common inherited bleeding disorder, affecting about 1% to 2% of people. Symptoms: ecchymosis, prolonged bleeding, oral bleeding, menorrhagia

9 vonwillebrand Project Qualitative and/or quantitative deficiency of FvW (vonwillebrand factor) Important role in the haemeostasis processes Platelet aggregation and adhesion

10 vonwillebrand Project Variability in the Symptomatology (type and severity) Six categories with different therapeutic requirements Traditional Diagnosis Clinical history, bleeding time measurement, analysis of global platelet function, FVW activity as a cofactor of ristocetin, antigen quantification Laborious evaluation, high economic cost. Not always it is possible to establish a trustworthy diagnosis

11 vonwillebrand Project Importance of Molecular Diagnosis of vw disease Improve the Diagnosis (reduce false negatives or positives) Defective FvW / Low levels FvW Administration of external FvW Stimulation of the FvW production Gene with some complexity. Within an autosomic chromosome. Partial Pseudogene in chr p kb 52 exons 9 kb mrna 2813 Amino Acids

12 vonwillebrand Project Goal Molecular characterization of vw patients using NGS 200 samples x 178 kb = 35,6 Mb + relatives It is very difficult to do it by Sanger Sequencing Easly sequenced by GA Illumina 1 run: 6 40 Gb

13 vonwillebrand Project 15 Long Range PCRs amplification of the entire gene DNA fragmentation using a biorutor Library preparation using an indexing system Single-Read sequencing runs of 36 cycles

14 vonwillebrand Project Multiplex System Adapters 5 -ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3 PCR Primers TGGAC 5 -AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC-3 5 -CAAGCAGAAGACGGCATACGAGCTCTTCCGATC-3 Sequencing Primer 5 -ACACTCTTTCCCTACACGACGCTCTTCCGATC-3

15 vonwillebrand Project 10 x 178 Kb = 1,78 Mb Sample 1 x 36 x x 100 = 360 Mb Lane 360 / 1, x Coverage With the new versions of GA and reagents: clusters/tile 1 Gb/lane Need for an intermediate capacity sequencer

16 vonwillebrand Project * All the known mutations were found * New mutations were found Mutations Different categories of vw disease Diagnosis kit

17 7q32 Project University of Navarra (CIMA) - Progenika SMZL Splenic Marginal Zone Lymphoma Features * Splenomegaly * Infiltration of bone marrow and peripheral blood in the spleen (perhaps also in lymph nodes and liver)

18 7q32 Project 80 % SMZL patients have chromosomal abnormalities trisomies, deletions, duplications, translocations 7q32 5 Mb x 7 patients = 35 Mb NGS - Identification of the tumor suppressor gene (or genes) in the 7q32 region

19 7q32 Project DNA fragmentation using a biorutor Library preparation with sequence capture Single-Read sequencing run of 36 cycles SureSelect from Agilent In solution capture

20 7q32 Project Results: 107 SNPs in exons (64% annotated) 60% of SNPs were validated Interestings SNPs: -With aa. Change -New SNP, not annotated -Could be validated

21 Illumina Genome Analyzer Progenika Experience. Resequecing. de novo Sequencing. small RNA. ChIP-Seq. mrna