Dataset Information

A preliminary evaluation of next-generation sequencing as a screening tool for targeted genotyping of erythrocyte and platelet antigens in blood donors.

ABSTRACT: BACKGROUND: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. MATERIALS AND METHODS: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. RESULTS: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. DISCUSSION: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.

SUBMITTER: Orzinska A

PROVIDER: S-EPMC5919841 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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A preliminary evaluation of next-generation sequencing as a screening tool for targeted genotyping of erythrocyte and platelet antigens in blood donors.

Orzińska Agnieszka A Guz Katarzyna K Mikula Michał M Kulecka Maria M Kluska Anna A Balabas Aneta A Pelc-Kłopotowska Monika M Ostrowski Jerzy J Brojer Ewa E

Blood transfusion = Trasfusione del sangue 20170310 3

<h4>Background</h4>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 ...[more]

PMID: 28287381

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Project description:BackgroundTargeted next-generation sequencing (NGS) enables rapid identification of common and rare genetic variation. The detection of variants contributing to therapeutic drug response or adverse effects is essential for implementation of individualized pharmacotherapy. Successful application of short-read based NGS to pharmacogenes with high sequence homology, nearby pseudogenes and complex structure has been previously shown despite anticipated technical challenges. However, little is known regarding the utility of such panels to detect copy number variation (CNV) in the highly polymorphic cytochrome P450 (CYP) 2D6 gene, or to identify the promoter (TA)7 TAA repeat polymorphism UDP glucuronosyltransferase (UGT) 1A1*28. Here we developed and validated PGxSeq, a targeted exome panel for pharmacogenes pertinent to drug disposition and/or response.MethodsA panel of capture probes was generated to assess 422 kb of total coding region in 100 pharmacogenes. NGS was carried out in 235 subjects, and sequencing performance and accuracy of variant discovery validated in clinically relevant pharmacogenes. CYP2D6 CNV was determined using the bioinformatics tool CNV caller (VarSeq). Identified SNVs were assessed in terms of population allele frequency and predicted functional effects through in silico algorithms.ResultsAdequate performance of the PGxSeq panel was demonstrated with a depth-of-coverage (DOC) ≥ 20× for at least 94% of the target sequence. We showed accurate detection of 39 clinically relevant gene variants compared to standard genotyping techniques (99.9% concordance), including CYP2D6 CNV and UGT1A1*28. Allele frequency of rare or novel variants and predicted function in 235 subjects mirrored findings from large genomic datasets. A large proportion of patients (78%, 183 out of 235) were identified as homozygous carriers of at least one variant necessitating altered pharmacotherapy.ConclusionsPGxSeq can serve as a comprehensive, rapid, and reliable approach for the detection of common and novel SNVs in pharmacogenes benefiting the emerging field of precision medicine.

Project description:Genotyping of the human leucocyte antigen (HLA) is indispensable for various medical treatments. However, unambiguous genotyping is technically challenging due to high polymorphism of the corresponding genomic region. Next-generation sequencing is changing the landscape of genotyping. In addition to high throughput of data, its additional advantage is that DNA templates are derived from single molecules, which is a strong merit for the phasing problem. Although most currently developed technologies use genomic DNA, use of cDNA could enable genotyping with reduced costs in data production and analysis. We thus developed an HLA genotyping system based on next-generation sequencing of cDNA.Each HLA gene was divided into 3 or 4 target regions subjected to PCR amplification and subsequent sequencing with Ion Torrent PGM. The sequence data were then subjected to an automated analysis. The principle of the analysis was to construct candidate sequences generated from all possible combinations of variable bases and arrange them in decreasing order of the number of reads. Upon collecting candidate sequences from all target regions, 2 haplotypes were usually assigned. Cases not assigned 2 haplotypes were forwarded to 4 additional processes: selection of candidate sequences applying more stringent criteria, removal of artificial haplotypes, selection of candidate sequences with a relaxed threshold for sequence matching, and countermeasure for incomplete sequences in the HLA database.The genotyping system was evaluated using 30 samples; the overall accuracy was 97.0% at the field 3 level and 98.3% at the G group level. With one sample, genotyping of DPB1 was not completed due to short read size. We then developed a method for complete sequencing of individual molecules of the DPB1 gene, using the molecular barcode technology.The performance of the automatic genotyping system was comparable to that of systems developed in previous studies. Thus, next-generation sequencing of cDNA is a viable option for HLA genotyping.

Dataset Information

A preliminary evaluation of next-generation sequencing as a screening tool for targeted genotyping of erythrocyte and platelet antigens in blood donors.

Publications

A preliminary evaluation of next-generation sequencing as a screening tool for targeted genotyping of erythrocyte and platelet antigens in blood donors.

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