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Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the ACKR1 Gene Encoding the Duffy Antigens.

ABSTRACT: Background:In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. Materials and Methods:After long-range PCR amplification spanning the whole ACKR1 gene locus (?2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. Results:High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. Discussion:Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a "longitudinal" overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.

SUBMITTER: Fichou Y

PROVIDER: S-EPMC7036534 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the <i>ACKR1</i> Gene Encoding the Duffy Antigens.

Fichou Yann Y Berlivet Isabelle I Richard Gaëlle G Tournamille Christophe C Castilho Lilian L Férec Claude C

Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie 20191211 1

<h4>Background</h4>In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of <i>ACKR1</i>, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach.<h4>Materials ...[more]

PMID: 32110191

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Project description:Regulatory guidance documents stress the value of assessing the most appropriate endpoints in multiple tissues when evaluating the in vivo genotoxic potential of chemicals. However, conducting several independent studies to evaluate multiple endpoints and/or tissue compartments is resource intensive. Furthermore, when dependent on visual detection, conventional approaches for scoring genotoxicity endpoints can be slow, tedious, and less objective than the ideal. To address these issues with current practices we attempted to (1) devise resource sparing treatment and harvest schedules that are compatible with liver and blood micronucleus endpoints, as well as the Pig-a gene mutation assay, and (2) utilize flow cytometry-based methods to score each of these genotoxicity biomarkers. Proof-of-principle experiments were performed with 4-week-old male and female Crl:CD(SD) rats exposed to aristolochic acids I/II, benzo[a]pyrene, cisplatin, cyclophosphamide, diethylnitrosamine, 1,2-dimethylhydrazine, dimethylnitrosamine, 2,6-dinitrotoluene, hydroxyurea, melphalan, temozolomide, quinoline, or vinblastine. These 13 chemicals were each tested in two treatment regimens: one 3-day exposure cycle, and three 3-day exposure cycles. Each exposure, blood collection, and liver harvest was accomplished during a standard Monday-Friday workweek. Key findings are that even these well-studied, relatively potent genotoxicants were not active in both tissues and all assays (indeed only cisplatin was clearly positive in all three assays); and whereas the sensitivity of the Pig-a assay clearly benefitted from three versus one treatment cycle, micronucleus assays yielded qualitatively similar results across both study designs. Collectively, these results suggest it is possible to significantly reduce animal and other resource requirements while improving assessments of in vivo genotoxicity potential by simultaneously evaluating three endpoints and two important tissue compartments using fit-for-purpose study designs in conjunction with flow cytometric scoring approaches. Environ. Mol. Mutagen., 60:704-739, 2019. © 2019 Wiley Periodicals, Inc.

Dataset Information

Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the ACKR1 Gene Encoding the Duffy Antigens.

Publications

Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the <i>ACKR1</i> Gene Encoding the Duffy Antigens.

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