Development of bovine leukocyte antigen – DRB3 genotyping using nanopore sequencing

IF 2.1 3区 生物学 Q2 AGRICULTURE, DAIRY & ANIMAL SCIENCE
Animal genetics Pub Date : 2025-08-03 DOI:10.1111/age.70033
Takenori Arai, Yu-ichi Kanetsuna, Saki Miyake, Saki Uehara, Kazuya Nagai, Kei-ich Matsuda, Toh-ichi Hirata, Yusuke Sakai, Takashi Matsuzaki, Shinji Yamada, Hirokazu Hikono, Kenji Murakami
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Abstract

The bovine leukocyte antigen DRB3 (BoLA-DRB3) gene, a crucial component of major histocompatibility complex class (MHC) II, influences disease susceptibility and production traits in cattle. Conventional PCR – (Sanger)-sequence-based typing (PCR-SBT) – has limitations, including time-intensive processing and phase ambiguity issues. We developed a nanopore-sequencing-based method for BoLA-DRB3 genotyping. The full-length gene (10.9 kbp) was amplified from blood samples of 109 Japanese Black cattle and sequenced using Oxford Nanopore Technologies’ nanopore MinION. The method achieved high-quality metrics, with a mean coverage of 99.97%, an average sequencing depth of 2599× and a mean Q-score of 20.5. Perfect reference sequence alignment was obtained in 92.7% (101/109) of samples. Alleles of all samples showed complete concordance with the set of PCR-SBT typing results for exon 2. The entire process was completed within 3 days (6 h of hands-on time), substantially reducing the time and cost requirements compared with other methods. This nanopore sequencing method provides accurate BoLA-DRB3 genotyping while resolving phase ambiguity through full-length molecule analysis, presenting a potential new standard for bovine MHC genotyping.

Abstract Image

利用纳米孔测序技术建立牛白细胞抗原- DRB3基因分型
牛白细胞抗原DRB3 (BoLA-DRB3)基因是主要组织相容性复合体类(MHC) II的重要组成部分,影响牛的疾病易感性和生产性状。传统的PCR- (Sanger)-序列分型(PCR- sbt)-具有局限性,包括时间密集的处理和相模糊问题。我们开发了一种基于纳米孔测序的BoLA-DRB3基因分型方法。从109头日本黑牛的血液样本中扩增出全长基因(10.9 kbp),并使用牛津纳米孔技术公司的纳米孔MinION进行测序。该方法获得了高质量的指标,平均覆盖率为99.97%,平均测序深度为2599x,平均Q-score为20.5。92.7%(101/109)的样本获得了完美的对照序列比对。所有样本的等位基因均与外显子2的PCR-SBT分型结果完全一致。整个过程在3天(6小时的动手时间)内完成,与其他方法相比,大大减少了时间和成本要求。这种纳米孔测序方法提供了准确的BoLA-DRB3基因分型,同时通过全长分子分析解决了相歧义,为牛MHC基因分型提供了一种潜在的新标准。
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来源期刊
Animal genetics
Animal genetics 生物-奶制品与动物科学
CiteScore
4.60
自引率
4.20%
发文量
115
审稿时长
5 months
期刊介绍: Animal Genetics reports frontline research on immunogenetics, molecular genetics and functional genomics of economically important and domesticated animals. Publications include the study of variability at gene and protein levels, mapping of genes, traits and QTLs, associations between genes and traits, genetic diversity, and characterization of gene or protein expression and control related to phenotypic or genetic variation. The journal publishes full-length articles, short communications and brief notes, as well as commissioned and submitted mini-reviews on issues of interest to Animal Genetics readers.
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