Estimates of Sequences with Ultralong and Short CDR3s in the Bovine IgM B Cell Receptor Repertoire Using the Long-read Oxford Nanopore MinION Platform.

Q3 Medicine
Tess E Altvater-Hughes, Harold P Hodgins, Douglas C Hodgins, Natasha B Gallo, Gabhan I Chalmers, Nicole D Ricker, Bonnie A Mallard
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Abstract

Cattle produce Abs with an H chain ultralong CDR3 (40-70 aa). These Abs have been shown to have features such as broad neutralization of viruses and are investigated as human therapeutics. A common issue in sequencing the bovine BCR repertoire is the sequence length required to capture variable (V) and isotype gene information. This study aimed to assess the use of Oxford Nanopore Technologies' MinION platform to perform IgM BCR repertoire sequencing to assess variation in the percentage of ultralong CDR3s among dairy cattle. Blood was collected from nine Holstein heifers. B cells were isolated using magnetic bead-based separation, RNA was extracted, and IgM+ transcripts were amplified using PCR and sequenced using a MinION R10.4 flow cell. The distribution of CDR3 lengths was trimodal, and the percentage of ultralong CDR3s ranged among animals from 2.32 to 20.13% in DNA sequences and 1.56% to 17.02% in productive protein sequences. V segment usage varied significantly among heifers. Segment IGHV1-7, associated with ultralong CDR3s, was used in 5.8-24.2% of sequences; usage was positively correlated with ultralong CDR3 production (r = 0.99, p < 0.01). To our knowledge, this is the first study to sequence the bovine BCR repertoire using Oxford Nanopore Technologies and demonstrates the potential for cost-efficient long-read repertoire sequencing in cattle without assembly. Findings from this study support literature describing the distribution of length and percentage of ultralong CDR3s. Future studies will investigate changes in the bovine BCR repertoire associated with age, antigenic exposure, and genetics.

使用长读数牛津纳米孔 MinION 平台估算牛 IgM B 细胞受体序列中的超长和短 CDR3 序列。
牛产生的抗体具有 H 链超长 CDR3(40-70 aa)。这些抗体已被证明具有广泛中和病毒等特性,并被研究用作人类疗法。牛 BCR 基因库测序的一个常见问题是捕获可变 (V) 和同种型基因信息所需的序列长度。本研究旨在评估使用牛津纳米孔技术公司(Oxford Nanopore Technologies)的 MinION 平台进行 IgM BCR 复合物测序的效果,以评估奶牛中超长 CDR3 百分比的变化。采集九头荷斯坦小母牛的血液。使用磁珠分离法分离 B 细胞,提取 RNA,使用 PCR 扩增 IgM+ 转录物,并使用 MinION R10.4 流式细胞仪进行测序。CDR3长度的分布呈三足鼎立之势,超长CDR3在动物DNA序列中的比例从2.32%到20.13%不等,在生产性蛋白质序列中的比例从1.56%到17.02%不等。不同母牛的 V 段使用情况差异很大。5.8-24.2%的序列中使用了与超长CDR3相关的IGHV1-7片段;使用率与超长CDR3的产量呈正相关(r = 0.99,p<0.05)。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
3.70
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0.00%
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审稿时长
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