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Massively parallel and multiplex blood group genotyping using next-generation-sequencing
Affiliation:1. AXO Science SAS, 66 Bd Niels Bohr CEI 1, 69100 Villeurbanne, France;2. ViroScan3D SAS, 11 allée des acacias, 01600 Trévoux, France;3. ProfileXpert SFR-Santé Lyon Est, UCBL UMS, 3453 CNRS-US7 INSERM, Lyon, France;4. Univ Lyon, Université Lyon1CNRS, INSA, CPE-Lyon, ICBMS, UMR 5246, 43, Bd du 11 novembre 1918, 69622 Villeurbanne cedex, France;5. Université de Lyon, Université Claude Bernard Lyon1, 43, Bd du 11 novembre 1918, 69622 Villeurbanne cedex, France;6. CRCL Cancer Research center of LYON, INSERM U1052, CNRS UMR5286, Centre Lyon Bérard, Lyon, France;7. CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS UMR5308, ENS de Lyon, Université Lyon, Hospices Civils de Lyon, Lyon, France;8. Ecologie Microbienne, CNRS UMR5557, Université Claude Bernard Lyon 1, Lyon, France;1. Division of Clinical Pathology/Laboratory Medicine, Department of Pathology, Montefiore Medical Center, Bronx, NY;2. Department of Transfusion Medicine, NIH Clinical Center, National Institutes of Health, Bethesda, MD;1. Department of Technology, Faculty of Health and Technology, University College Copenhagen, Denmark;2. Department of Clinical Biochemistry, Hvidovre Hospital, Copenhagen University Hospital, Denmark;1. Institute of Education and Research of Santa Casa de Belo Horizonte Hospital - IEP/SCBH, Belo Horizonte, MG, Brazil;2. Neurosurgery Service - Santa Casa de Belo Horizonte Hospital, Belo Horizonte, MG, Brazil;3. Department of Pharmacology, Institute of Biosciences of Botucatu, Universidade Estadual Paulista (UNESP), São Paulo, Brazil;1. Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil;2. Institute of Tropical Medicine, Department of Infectious Disease, University of São Paulo, São Paulo, Brazil;3. Discipline of Hematology, University of São Paulo School of Medicine, São Paulo, Brazil;4. Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo school of Medicine
Abstract:ObjectivesThirty-six blood group systems are listed by the International Society of Blood Transfusion, containing almost 350 antigens. Most of these result from a single nucleotide polymorphism (SNP). Serology is the standard method for blood group typing. However, this technique has some limitations and cannot respond to the growing demand of blood product typing for a large number of antigens. Here we describe a blood group genotyping assay directly from whole blood samples using Next-Generation Sequencing (NGS), allowing the simultaneous identification of 15 SNPs associated with the blood group systems of 95 patients in a single run.Design and methodAfter an automated DNA extraction, targets are amplified by multiplex polymerase chain reaction (PCRm). Two panels addressing 9 groups have been developed (MNS, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, and Colton), one for 8 SNPs, the other for 7 SNPs. For each sample, both panels corresponding to 14 amplicons (1 amplicon containing 2 SNPs) are pooled. Then a dual-indexed library is generated from each pool by linking Illumina adaptors directly onto amplicons, followed by sequencing using the MiSeq platform (Illumina).ResultsIn a single experiment, 95 blood donor samples have been sequenced for the genes of interest. Among the 1425 targeted single nucleotide polymorphisms, 1420 were identified by sequencing, reflecting a coverage of 99.65%. The obtained data shows a good correlation (99% for all SNPs) with other blood group typing methods. Depending on the allele pairs analyzed, correlations vary between 97.12 and 100%.Conclusion: Next-Generation sequencing would supplement serological and molecular techniques and, in the near future, could replace it with complete and fast results acquisition for pre-screening and identification of rare blood bags.
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