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Next Generation Sequencing: Application to Transfusion M edicine and I mmunohematology ?

Next Generation Sequencing: Application to Transfusion M edicine and I mmunohematology ?. O. Preynat-Seauve Laboratory of immunohematology Hematology Unit Laboratory medicine unit Geneva University Hospital olivier.preynat-seauve@hcuge.ch. DNA and RNA sequencing.

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Next Generation Sequencing: Application to Transfusion M edicine and I mmunohematology ?

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  1. NextGeneration Sequencing: Application to Transfusion Medicineand Immunohematology? O. Preynat-Seauve Laboratory of immunohematology Hematology Unit Laboratorymedicine unit Geneva UniversityHospital olivier.preynat-seauve@hcuge.ch

  2. DNA and RNA sequencing “the process of determining the precise order of nucleotides within a nucleic acid molecule” DNA RNA Plants Microbes Human/animal cells and tissues Vaccines ... Blood products ...

  3. The history of sequencing 1977: « Maxam Gilbert Sequencing » 2013: « nextgenerationmethods » or « highthroughput sequencing » >500 000 sequencing operationscanberunned in parrallel WHOLE genome, transcriptome , miRNome etc. Only fragments 

  4. NextGeneration Sequencing (NGS): variousmethods

  5. The mostwidelyused system isprovided by the Illumina company “the simultaneous sequencing of millions of tiny fragments of DNA on the surface of a glass slide about the size of a large matchbox”

  6. The machine produces millions of short sequencescalled « READS » Millions of reads ATGG...CGCA TTGA...ATGCG TATA....CTA GGC...AATAA etc. etc. Reads (= fragments) are reasembled by softwares into « CONTIGS » TTGA...ATGCGGGC...AATAAATGG...CGCA CONTIGS are identifiedusingdatabases (bioinformatics) each portion of the genome/RNome is represented multiple times in different fragment frames (fragmentation is at random) Genome position

  7. Wholegenome/transcriptomesequencing: interest for immunohematology and transfusion medicine ?

  8. Wholesequencingfor immunohematology ? • Single analysis of the entireblood groups genotype • Determination of a global profile in one step • Exhaustive identification of blood groups variants, rare genotypes etc. • Targets ? Blood groups antigens, HLA, minorantigens

  9. * Tooheavy /expensive/slow as compared to existingmethods? * Less quantitative than PCR ? * Sensitivity ? * False positive/false negativerates? (and controls for eachgene!) * Can weeasilydeduce the phenotypefrom the genotype ?

  10. To technicallysequence a wholegenomeiscurrently « easy » and not to muchexpensive … and finallyyouobtain a CD with millions and millions of data Remark: do not start if you do not have in your team a bioinformatician! sequencing (2 weeks) analysis (months, years!)

  11. Interest of sequencing for transfusion medicine? • Landscape of nucleicacidspresent in bloodproducts ? • The completenucleicacid content in bloodproductsis not known Blood product Nucleicacidsassociatedwithresidualleukocytes Cell-free nucleicacids Nucleicacidassociatedwithcells (redbloodcells or platelets)

  12. Landscape of non-humannucleicacids in bloodproducts ? • All the virusesthat « escape » to bloodproducts qualification: • Emergent viruses ? • Inocuousviruses(thatcould have impact on immunocompromisedpatients) • Otherinfectious agents signatures ? Freshfrozen plasma Redbloodcellsconcentrate Plateletsconcentrate Reinforcment (or not) of pathogensinactivation ? Additonal virus testing for immunocompromised patients ?

  13. Development of a bioinformaticsoftwarefor virus screen in a wholeRNA sequence (Illumina) Specificity Pos. controls Assemblies (CONTIGS) Dr Thomas Petty, postdoc

  14. Pipeline validation using CMV/Sendaï virus-infectedcells Dr Erika Cosset, postdoc Dr Thomas Petty, postdoc

  15. neg. control Virus-free samples (glioblastoma) neuroepithelialcells neuroepithelialcells+CMV neuroepithelialcells+Sendaï virus Percent of the virus genomethatiscovered by reads = GENOME COVERAGE Number of matchingreads

  16. This binarycomputationalanalysismixinggenomecoverageand number of readsprovideuseful informations in thiscontext of virus discovery High virus replication Low virus replication Latent Latent virusesreactivatingsomegeneswithout virions replication (CMV) No virions/viral genereactivation

  17. Ongoingproject: virus screen in bloodproducts 10 pools of 10 plasma unit samples ( 100 donors) 10 pools of 10 redbloodcells unit samples ( 100 donors) Negativecontrols (buffer alone) Positive controls: bloodproductssamplesinfected by CMV/Sendaï virus DNA seq RNA seq Bioinformatic pipeline Exhaustive « picture »of the virologicalstatus of bloodproducts

  18. Landscape of humannucleicacids in bloodproducts ?

  19. CELLS mRNA (haemoglobin !) rRNA tRNA miRNA mitDNA residual plasma Cell-free nucleicacids plasma Cell-free nucleicacids Residualleukocytes Genomic/mitochondrial DNA all RNAs Microparticles miRNA Redbloodcells plasma platelets

  20. Cell-free nucleicacids (plasma) • ds short DNA (70-200 base pair) • ds long DNA (< 21 kb) • mRNA • miRNA (very active !) • NeutrophilExtracellularTraps (NETs) • Sources: cellnecrosis, apoptosis, active secretion (lymphocytes, neutrophils) • Nucleicacidspresent in microparticles BIOLOGICAL ACTIVITY IN RECIPIENT ?

  21. NGS and transfusion: concludingremarks • research: provide a new tool to improve the knowledge of transfusion and immmunohematology • routine: Potentialinterest in the future ??

  22. Laboratory of immunohematology Geneva UniversityHospital Erika Cosset Thomas Petty Olivier Preynat-Seauve ARTERES Foundation, Geneva ISREC Foundation, Lausanne Egon NaefFoundation, Geneva Department of Genetic and LaboratoryMedicine Laboratory of Virology Geneva UniversityHospital Laurent Kaiser Samuel Cordey Oncology Unit Geneva UniversityHospital Pierre-Yves Dietrich Valérie Dutoit Swiss Institute of Bioinformatic EvgenyZbodnov IsmelPalladieau GenomicCoreFacility Faculty of medicine Geneva FASTERIS SA, Plan-Les-Ouates Blood Transfusion Center Geneva UniversityHospital Emanuel Rigal Soraya El-Dusouqui Hematology Unit Geneva UniversityHospital Thomas-Pierre Lecompte

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