长角血蜱饥饿雌成蜱转录组测序及分析
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摘要
目的利用IlluminaHiSeq高通量技术对长角血蜱饥饿雌成蜱进行转录组测序。方法将测序得到的数据进行拼接组装,并利用生物信息学方法对所得序列进行基因功能注释、功能分类、代谢途径分析和简单重复序列标记等分析。结果共获得181 246 184个clean reads数据,组装后得到107 428个unigenes序列,平均长度1 246.29。以BLAST软件将所有的unigenes序列与Nr,Nt,Pfam,KOG,Swiss-prot,KEGG,GO数据库进行比对。与Nr数据库比对发现长角血蜱基因序列与同科肩突硬蜱(Ixodes scapularis)具有较高的同源性,为55.3%;根据GO数据库的注释,将其分为生物过程、细胞组分和分子功能3大类共56分支;通过与KOG数据库的注释划分25类;根据与KEGG数据库的分析发现含有代谢通路相关的基因有32类,其中较多的是信号转导(signal transduction)类,占12.13%。通过SSR位点查找共鉴定出45 863个SSR;SNP分析发现发生转换的SNP数为195 369个,发生颠换的SNP数为96 780个。结论通过对长角血蜱饥饿雌成蜱转录组水平分析,可为后续长角血蜱功能基因的挖掘及表达谱等方面的研究奠定基础。
Objective To sequence transcriptomes of unfed female Haemaphysalis longicornis using IlluminaHiSeq highthroughput technology. Methods The data on sequences the transcriptomes were spliced and assembled, and the obtained sequences were analyzed with functional annotation, functional classification, metabolic pathway analysis and simple repeated sequence markers using bioinformatics methods. Results A total of 181 246 184 clean reads data were obtained and 107 428 unigene sequences were obtained after assembly, with an average length of 1 246.29. All unigene sequences were aligned with the Nr, Nt, Pfam, KOG, Swiss-prot, KEGG, GO databases using BLAST software. Compared with the Nr database, the long-horned blood scorpion gene sequence has a high homology(55.3%) with that of Ixodes scapularis.According to the annotation of the GO database, the functions of the all unigene sequences were divided into 3 categories(biological process, cellular component and molecular function) covering 56 functional groups; based on to the annotations of the KOG database, all the unigene sequences were assigned into 25 categories; while, according to the analysis of the KEGG database, there are 32 groups of genes involved in metabolic pathways and a major part of them(12.13%) are involved in signal transduction. A total of 45 863 simple sequence repeats(SSRs) were identified with SSR locus search. Single nucleotide polymorphism(SNP) analysis indicated that the number of SNPs for base transition was 195 369 and that for base transversion was 96 780. Conclusion The analysis on transcriptomes of unfed female Haemaphysalis longicornis lays a foundation for subsequent researches on gene expression and expression of the tick.
Objective To sequence transcriptomes of unfed female Haemaphysalis longicornis using IlluminaHiSeq highthroughput technology. Methods The data on sequences the transcriptomes were spliced and assembled, and the obtained sequences were analyzed with functional annotation, functional classification, metabolic pathway analysis and simple repeated sequence markers using bioinformatics methods. Results A total of 181 246 184 clean reads data were obtained and 107 428 unigene sequences were obtained after assembly, with an average length of 1 246.29. All unigene sequences were aligned with the Nr, Nt, Pfam, KOG, Swiss-prot, KEGG, GO databases using BLAST software. Compared with the Nr database, the long-horned blood scorpion gene sequence has a high homology(55.3%) with that of Ixodes scapularis.According to the annotation of the GO database, the functions of the all unigene sequences were divided into 3 categories(biological process, cellular component and molecular function) covering 56 functional groups; based on to the annotations of the KOG database, all the unigene sequences were assigned into 25 categories; while, according to the analysis of the KEGG database, there are 32 groups of genes involved in metabolic pathways and a major part of them(12.13%) are involved in signal transduction. A total of 45 863 simple sequence repeats(SSRs) were identified with SSR locus search. Single nucleotide polymorphism(SNP) analysis indicated that the number of SNPs for base transition was 195 369 and that for base transversion was 96 780. Conclusion The analysis on transcriptomes of unfed female Haemaphysalis longicornis lays a foundation for subsequent researches on gene expression and expression of the tick.
引文
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[13]Davidson NM,Oshlack A.Corset:enabling differential gene expression analysis for de novoassembled transcriptomes[J].Genome Biology,2014,15(7):410.
[14]Dillies MA,Rau A,Aubert J,et al.A comprehensive evaluation of normalization methods for Illumina high-throughput RNAsequencing data analysis[J].Briefings in Bioinformatics,2013,14(6):671-683.
[2]Jiang XL,Wang XJ,Li JD,et al.Isolation,identification and characterization of SFTS bunyavirus from ticks collected on the surface of domestic animals[J].Chinese Journal of Virology,2012,28(3):252-257.
[3]Park SW,Song BG,Shin EH,et al.Prevalence of severe fever with thrombocytopenia syndrome virus in Haemaphysalis longicornis ticks in South Korea[J].Ticks and Tick-Borne Diseases,2014,5(6):975-977.
[4]张臣臣,张仪.我国常见蜱的生物学特性及其传播疾病研究进展[J].国际医学寄生虫病杂志,2015,42(3):184-188.
[5]Chen X,Xu S,Yu Z,et al.Multiple lines of evidence on the genetic relatedness of the parthenogenetic and bisexual Haemaphysalis longicornis(Acari:Ixodidae)[J].Infection,Genetics and Evolution,2014,21:308-314.
[6]孙文英.长角血蜱孤雌生殖种群防御素基因克隆、原核表达及功能分析[D].河北师范大学,2016.
[7]赵华渠.长角血蜱唾液腺功能蛋白发掘与吸血适应机制研究[D].河北师范大学,2017.
[8]刘建刚,刘光远,田占成,等.长角血蜱雄蜱抑制消减杂交cDNA文库的构建与分析[J].中国兽医科学,2009,39(8):659-664.
[9]da Silva Vaz I Jr,Torino Lermen T,Michelon A,et al.Effect of acaricides on the activity of a Boophilus microplus glutathione S-transferase[J].Veterinary Parasitology,2004,119(2):237-245.
[10]Josek T,Walden KKO,Allan BF,et al.A foreleg transcriptome for Ixodes scapularis ticks:candidates for chemoreceptors and binding proteins that might be expressed in the sensory Haller′s organ[J].Ticks and Tick-borne Diseases,2018.
[11]Maher CA,Kumar-Sinha C,Cao XH,et al.Transcriptome sequencing to detect gene fusions in cancer[J].Nature,2009,458(7234):97-101.
[12]Grabherr MG,Haas BJ,Yassour M,et al.Full-length transcriptome assembly from RNA-Seq data without a reference genome[J].Nature Biotechnology,2011,29(7):644.
[13]Davidson NM,Oshlack A.Corset:enabling differential gene expression analysis for de novoassembled transcriptomes[J].Genome Biology,2014,15(7):410.
[14]Dillies MA,Rau A,Aubert J,et al.A comprehensive evaluation of normalization methods for Illumina high-throughput RNAsequencing data analysis[J].Briefings in Bioinformatics,2013,14(6):671-683.
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