基于微量RNA高通量测序技术的牦牛MⅡ期卵母细胞转录组研究  

Transcriptome Analysis of Yak Metaphase Ⅱ(MⅡ) Oocytes by a Micro-transcriptome Sequencing Method

作　　者：兰道亮[1] 熊显荣[2] 林宝山[2] 陈亚冰[2] 胡敏 苏小珊 李键 

机构地区：[1]西南民族大学青藏高原研究院,成都610041 [2]西南民族大学生命科学与技术学院,成都610041 [3]深圳华大基因研究院,深圳518083

出　　处：《畜牧兽医学报》2014年第5期722-732,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技支撑计划(2012BAD13B06);国家公益性行业(农业)科研专项(201203009)

摘　　要：旨在现有高通量测序的基础上,建立一种针对牦牛卵母细胞等微量样本的RNA高通量测序方法,为进一步研究牦牛卵母细胞的转录组学提供基础。以微量MII期牦牛卵母细胞RNA(10ng)作为研究样本,应用SMART cDNA PCR扩增技术对样本进行富集并构建测序文库,再应用改进的RNA高通量测序技术对其进行高通量测序分析。经Illumina-Solexa深度测序后,得到了一个包含47 619 254条测序序列,4Gb大小数据量的微量MII期牦牛卵母细胞测序文库。质量控制(Quality control,QC)及基本结果分析表明测序文库质量良好。基因组比对分析显示,共有15 626个牦牛基因被映射。此外,还获得了7 348个新的转录本。GO分类注释结果显示,共有13 795个比对上的基因参与了生物过程、细胞组分及分子功能3大主要类别。进一步富集分析显示,分别有333 134及113个GO类别在上述3大类中得到富集。KEGG通路分析结果显示,共有13 496个比对上的基因参与了258条通路,其中101条通路得到有效富集。本研究以牦牛卵母细胞为样本成功建立了一种起始RNA量可低至10ng的微量转录组测序方法。该研究结果为进一步研究牦牛基因组提供了基础,同时也为研究牦牛MII期卵母细胞的分子机理提供了新的视角。The aim of study was established an improved micro-transcriptome sequencing method by using yak oocytes as the trace samples,and provided the basis for the further transcriptome study of yak oocyte.The micro-transcriptome sequencing library were constructed based on the SMART PCR-amplified cDNAs technology by using yak oocytes as the model cell with a starting amount of total RNA as low as 10ng RNA,and then an improved high-throughput(HT)sequencing technology were applied for transcriptome analysis.After Illumina-Solexa deep sequencing,a total of 47 619 254clean reads with 4Gb data were obtained in the micro-oocyte library.Quality control(QC)tests and basic analysis results showed that the sequencing quality of the librarywas good.The alignment analysis results of the reference genome showed that a total of 15 626 genes from yaks were mapped.Additionally,7 348novel transcripts that could be positioned back to the yak genome were obtained.The GO analysis results revealed that 13 795mapped genes took part in three main categories,namely,biological processes,cellular components and molecular functions.Further enrichment analysis results showed that a total of 333 134and 113GO categories were enriched above the three main categories.The KEGG analysis results showed that 13 496mapped genes were involved in 258pathways,and the enrichment analysis results showed that a total of 101pathways were enriched.This study established an improved micro-transcriptome sequencing method with yak oocytes as a sample having as low as 10ng of total RNA.The data not only revealed useful information for future studies on yak genomes,but also provided new insights for the molecular mechanism of the MII stage of the yak oocytes.

关 键 词：牦牛 卵母细胞 MII期 转录组 微量 高通量测序 

分 类 号：S823[农业科学—畜牧学]