摘要
【目的】本研究旨在利用Oxford Nanopore测序技术组装和注释东方蜜蜂微孢子虫Nosema ceranae的高质量全长转录组。【方法】采用Nanopore PromethION系统对东方蜜蜂微孢子虫的纯净孢子进行转录组测序。通过识别每条clean read两端引物鉴定全长转录本序列。利用Blast工具将全长转录本比对Nr,Swiss-Prot,KOG,eggNOG,Pfam,GO和KEGG数据库,获得相应注释信息。分别利用蛋白结构域分析方法CPC,CNCI,CPAT和Pfam对长链非编码RNA(long noncoding RNA,lncRNA)进行预测,获得高可信度lncRNA。利用CPM(counts per million)法计算每一条全长转录本的表达量。【结果】利用Nanopore PromethION系统对东方蜜蜂微孢子虫转录组测序共测得6988795条raw reads,经质控获得6953469条clean reads,其中包含5143999条全长转录本。共鉴定到10243条非冗余全长转录本,N50和平均读长分别为1042 bp和894 bp,最大读长为4855 bp。有9342,4038,4283,2569,4859和3450条全长转录本分别注释到Nr,KOG,eggNOG,Pfam,GO和KEGG数据库。注释到东方蜜蜂微孢子虫、蜜蜂微孢子虫Nosema apis和家蚕微孢子虫Nosema bombycis的全长转录本数量最多。共鉴定到87条高可信度lncRNA,包含49条正义链lncRNA(sense lncRNA)、25条反义链lncRNA(anti-sense lncRNA)和13条基因间区lncRNA。本研究的测序量足以检测到全部表达的全长转录本,全长转录本的表达量(CPM)范围在0.1到10000以上。【结论】本研究构建和注释了东方蜜蜂微孢子虫的高质量全长转录组数据,可为病原的比较转录组分析、转录本的可变剪接和可变腺苷酸化分析、简单重复序列(simple sequence repeat,SSR)位点挖掘、基因结构优化以及基因全长序列克隆及功能研究提供关键基础。
【Aim】This study aims to assemble and annotate a high-quality full-length transcriptome of Nosema ceranae using Oxford Nanopore sequencing technology.【Methods】The transcriptome of clean spores of N.ceranae was sequenced using Nanopore PromethION system.Full-length transcripts were identified by recognizing primers at both ends of every clean read.Full-length transcripts were aligned to Nr,Swiss-Prot,KOG,eggNOG,Pfam,GO and KEGG databases to gain the corresponding annotations.Protein domain analysis methods including CPC,CNCI,CPAT and Pfam were used to predict long noncoding RNAs(lncRNAs),and the intersection was determined to be high-reliability lncRNAs.The expression level of each full-length transcript was calculated using CPM(counts per million)method.【Results】A total of 6988795 raw reads were obtained by Nanopore PromethION sequencing system,and 6953469 clean reads were gained after quality control,including 5143999 full-length transcripts.Besides,10243 non-redundant full-length transcripts were identified,with the N50,the average length and the maximum length of 1042,894 and 4855 bp,respectively.Furthermore,9342,4038,4283,2569,4859 and 3450 full-length transcripts were annotated to Nr,KOG,eggNOG,Pfam,GO and KEGG,respectively.Additionally,the majority of full-length transcripts were annotated to N.ceranae,Nosema apis and Nosema bombycis.Totally,87 high-reliability lncRNAs were identified,including 49 sense lncRNAs,25 anti-sense lncRNAs and 13 intergenic lncRNAs.The sequencing depth in this study was enough to detect all expressed full-length transcripts with the expression level(CPM)ranging from 0.1 to more than 10000.【Conclusion】The high-quality full-length transcriptome of N.ceranae was constructed and annotated in this study,laying a key foundation for comparative transcriptome analysis,investigation of alternative splicing and alternative adenylation of transcripts,identification of simple sequence repeat(SSR)loci,optimization of gene structure,and full-length sequence cloning and functional study of genes.
作者
陈华枝
杜宇
范小雪
祝智威
蒋海宾
王杰
范元婵
熊翠玲
郑燕珍
付中民
徐国钧
陈大福
郭睿
CHEN Hua-Zhi;DU Yu;FAN Xiao-Xue;ZHU Zhi-Wei;JIANG Hai-Bin;WANG Jie;FAN Yuan-Chan;XIONG Cui-Ling;ZHENG Yan-Zhen;FU Zhong-Min;XU Guo-Jun;CHEN Da-Fu;GUO Rui(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,China;Apitherapy Research Institute,Fujian Agriculture and Forestry University,Fuzhou 350002,China)
出处
《昆虫学报》
CAS
CSCD
北大核心
2020年第12期1461-1472,共12页
Acta Entomologica Sinica
基金
国家现代农业产业技术体系建设专项资金项目(CARS-44-KXJ7)
福建省自然科学基金项目(2018J05042)
福建省教育厅中青年教师教育科研项目(JAT170158)
福建农林大学杰出青年科研人才计划项目(xjq201814)
福建农林大学科技创新专项基金项目(CXZX2017342,CXZX2017343)
福建省大学生创新创业训练计划项目(3165602032,3155006018)。