title: A preliminary evaluation of next-generation sequencing as a screening tool for targeted genotyping of erythrocyte and platelet antigens in blood donors.
abstract: Matching the compatibility of donor blood with the recipient's antigens prevents alloimmunisation. Next-generation sequencing (NGS) technology is a promising method for extensive blood group and platelet antigen genotyping of blood donors. It circumvents the limitations of detecting known alleles based on predefined polymorphisms and enables targeted sequencing on a massive scale. The aim of this study was to evaluate the NGS AmpliSeq application on the Ion Torrent platform as a screening tool for genotyping blood donors' erythrocyte/platelet antigens.
  
  
  
  Primers for regions encoding antigens RhD (exons 5, 7), Rhc, RhE/e, Fya/b, Jka/b, M/N, S/s, HPA-1, 2, 3, 5, 15 were designed with Ion AmpliSeq Designer with manual inclusion of RHCE*C primers. DNA libraries of 57 regular blood donors with determined phenotype/genotype (prepared using the Ion AmpliSeq Library Kit and 14 primer pairs) were sequenced on the Ion Torrent PGM using 316v2 chips and 200 bp chemistry.
  
  
  
  Sequencing was successful in all but the MN and HPA-5 regions. Mean sequencing coverage in one experiment was 4,606 reads, except for the RHCE*C region (mean 568 reads). NGS results agreed with the known phenotype/genotype of donors except in one phenotypically Fy(a+b-) case in whom FY*A/FY*B alleles were found. Reading rates for homozygotes were 97-100%, while they were around 50% for heterozygotes. NGS of RHD regions led to identification of mutations in two RhD negative donors.
  
  
  
  NGS can be performed as a screening test to determine erythrocyte/platelet antigens in blood donors. This method allowed testing of 48 donors for 14 features (200 bp long) with the depth of a few thousand reads simultaneously, and the estimation of natural chimerism or hemi/homozygotic status. NGS screening can be adjusted to the genetic background of a given tested population.
authors.affiliation: Department of Immunohaematology and Immunology of Transfusion Medicine, Institute of Haematology and Blood Transfusion, Warsaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Orzińska
authors.affiliation: Department of Immunohaematology and Immunology of Transfusion Medicine, Institute of Haematology and Blood Transfusion, Warsaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Guz
authors.affiliation: Department of Genetics, "Maria Sklodowska-Curie" Memorial Cancer Centre and Institute of Oncology, Warsaw, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Mikula
authors.affiliation: Medical Centre of Postgraduate Education, Department of Gastroenterology, Hepatology and Clinical Oncology, Warsaw, Poland.
authors.forename: Maria
authors.initials: M
authors.lastname: Kulecka
authors.affiliation: Department of Genetics, "Maria Sklodowska-Curie" Memorial Cancer Centre and Institute of Oncology, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kluska
authors.affiliation: Department of Genetics, "Maria Sklodowska-Curie" Memorial Cancer Centre and Institute of Oncology, Warsaw, Poland.
authors.forename: Aneta
authors.initials: A
authors.lastname: Balabas
authors.affiliation: Department of Immunohaematology and Immunology of Transfusion Medicine, Institute of Haematology and Blood Transfusion, Warsaw, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Pelc-Kłopotowska
authors.affiliation: Department of Genetics, "Maria Sklodowska-Curie" Memorial Cancer Centre and Institute of Oncology, Warsaw, Poland.|Medical Centre of Postgraduate Education, Department of Gastroenterology, Hepatology and Clinical Oncology, Warsaw, Poland.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Ostrowski
authors.affiliation: Department of Immunohaematology and Immunology of Transfusion Medicine, Institute of Haematology and Blood Transfusion, Warsaw, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Brojer
mesh_terms: D016824:Antigens, Human Platelet / Q000235:genetics*; D001789:Blood Group Antigens / Q000235:genetics*; D001792:Blood Platelets; D004912:Erythrocytes; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D011110:Polymorphism, Genetic
chemical_list: D016824:Antigens, Human Platelet; D001789:Blood Group Antigens