基于RNA-Seq技术的犏牛囊胚冷冻前后单核苷酸多态性和可变剪切分析

doi:10.3969/j.issn.1674-5817.2020.04.003

实验动物与比较医学 ›› 2020, Vol. 40 ›› Issue (4): 279-.DOI: 10.3969/j.issn.1674-5817.2020.04.003

基于RNA-Seq技术的犏牛囊胚冷冻前后单核苷酸多态性和可变剪切分析

郑杰¹, 张国忠¹, 胡艳梅¹, 伍学一¹, 杨宗富¹, 字向东²

1. 攀枝花市妇幼保健院生殖医学中心, 攀枝花 617000;
2. 西南民族大学动物科学国家民委重点实验室, 成都 610041

收稿日期:2019-12-18 出版日期:2020-08-25 发布日期:2020-11-23
作者简介:郑杰(1992—), 男, 动物遗传育种与繁殖学硕士, 研究方向: 人类辅助生殖和动物繁殖生物学与胚胎工程研究。E-mail: 1067672953@qq.com
基金资助:
国家重点研发计划项目(2018YFD0502303)

Single Nucleotide Polymorphism and Alternative Splicing Analysis between Fresh and Vitrified-thawed Crossbred Blastocysts of Yak via RNA-Seq

ZHENG Jie¹, ZHANG Guozhong¹, HU Yanmei¹, WU Xueyi^1, YANG Zongfu¹, ZI Xiangdong²

1. Center for Reproductive Medicine, Panzhihua Maternity and Child Health Care Hospital, Panzhihua 617000, China; 2. Key Laboratory of Animal Science of State Ethnic Affairs Commission, Southwest Minzu University, Chengdu 610041, China

Received:2019-12-18 Online:2020-08-25 Published:2020-11-23

摘要/Abstract

摘要： 目的对犏牛囊胚玻璃化冷冻前后的单核苷酸多态性(single nucleotide polymorphism，SNP)和可变剪切(alternative splicing，AS)进行比较分析。方法采用普通娟珊牛精子和牦牛卵母细胞，以体外授精(in vitro fertilization，IVF)的方式获得犏牛囊胚。提取犏牛新鲜囊胚和经玻璃化冷冻复苏后的犏牛冻融囊胚总RNA，构建文库并进行高通量测序。采用SAMtools-0.1.19和ASprofile软件分别进行SNP和AS分析。结果犏牛新鲜囊胚和冻融囊胚样本通过去低质量序列、去接头污染等过程后，分别得到51 099 116和54 192 358条待分析数据(Clean Reads)。新鲜囊胚和冻融囊胚分别检测到116 681和224 750个SNP位点。在新鲜囊胚转换型SNP中，C/T类型数量略多于A/G类型；但在冻融囊胚中，A/G类型数量略多于C/T类型；两样本颠换型SNP中，A/T所占比例最少。新鲜囊胚和冻融囊胚转换和颠换型SNP的比例分别为2.57和2.45。新鲜囊胚和冻融囊胚分别检测出49 388和65 241个AS事件，主要有5种AS类型，其中转录起始区域和转录结束区域AS所占比例最大。结论利用RNA-Seq技术对玻璃化冷冻前后犏牛囊胚SNP和AS进行比较分析，可为后续人类胚胎冷冻前后相关基因功能分析和挖掘提供有效数据和理论基础。

关键词: 犏牛, 囊胚, 玻璃化冷冻, 转录组测序, 单核苷酸多态性, 可变剪切

Abstract: Objective To compare and analyze single nucleotide polymorphism (SNP) and alternative splicing (AS) between the fresh and vitrified-thawed crossbred blastocysts of the yak. Methods The crossbred blastocysts were derived from yak oocytes in vitro fertilized with Jersey cattle spermatozoa. Total RNA was extracted from these fresh blastocysts (FRB) and vitrified-thawed blastocysts (VTB), and then amplified and sequenced via the Smart-Seq2 method to construct RNA libraries. SNP and AS were analyzed by SAMtools-0.1.19 and ASprofile software, respectively. Results A total of 51 099 116 and 54 192 358 clean reads were obtained from the FRB and VTB, respectively. The SNP numbers of FRB and VTB were 116 681 and 224 750, the number of C/T type was slightly higher than that of A/G type for the FRB transition types of SNP (Ts). However, the number of A/G type was slightly higher than that of C/T type for the VTB Ts, and the number of A/T type was the least for all transversion types of SNP (Tv). The value of Ts/Tv in FRB and VTB were 2.57 and 2.45, respectively. The AS events in FRB and VTB were 49 388 and 65 241, respectively. It mainly had five patterns of AS, of which the two largest proportions were transcription start site and transcription terminal site. Conclusion The results of comparison and analysis of SNP and AS between the fresh and vitrified-thawed crossbred blastocysts of the yak provide valid data and theoretical basis for the following related gene function analysis and mining.

Key words: Crossbred yak, Blastocyst, Vitrification, RNA-Seq, Single nucleotide polymorphism, Alternative splicing

中图分类号:

Q95-33

郑杰, 张国忠, 胡艳梅, 伍学一, 杨宗富, 字向东. 基于RNA-Seq技术的犏牛囊胚冷冻前后单核苷酸多态性和可变剪切分析[J]. 实验动物与比较医学, 2020, 40(4): 279-.

ZHENG Jie, ZHANG Guozhong, HU Yanmei, WU Xueyi, YANG Zongfu, ZI Xiangdong. Single Nucleotide Polymorphism and Alternative Splicing Analysis between Fresh and Vitrified-thawed Crossbred Blastocysts of Yak via RNA-Seq[J]. Laboratory Animal and Comparative Medicine, 2020, 40(4): 279-.

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基于RNA-Seq技术的犏牛囊胚冷冻前后单核苷酸多态性和可变剪切分析

Single Nucleotide Polymorphism and Alternative Splicing Analysis between Fresh and Vitrified-thawed Crossbred Blastocysts of Yak via RNA-Seq

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