Sequencing And Bioinformatics Analysis Of MRNA From Echinococcus Granulosus Protoscolex

Objective To reveal the transcriptomic information, biological characteristics of theEchinococcus granulosus protoscole，we used RNA-Seq technique in this study. Throughbioinformatics analysis of sequencing data, we can recognize the relationship between theparasite and host, it also can provide novel platform for the development of vaccinecandidates and drug targets, provided basis database for the control and eradication of hydatiddisease.Methods1. Brood capsules were collected aseptically from fertile E. granulosus cysts inthe livers of an infectedpatient, then protoscoleces were isolated. Total RNA was isolatedfrom Echinococcus granulosus protoscolex using TRIzol.2. mRNA kit with Oligo (dT)magnetic beads was used to isolate poly(A) mRNA after total RNA was collected from E.granulosus protoscolex, the total mRNA was no less than500ng，then the total mRNA wasreversed transcription into cDNA. Fragmentation buffer was added to interrupt mRNA toshort fragments.Following the agarose gel electrophoresis, a range of cDNA fragments wereexcised from the gel, and selected for the PCR amplification as templates using kit.3. lluminaHiSeq2000was applied to sequencing.4. Prior to assembly, the high-quality clean readsobtained from raw data by removing adaptor sequences, duplication sequences. Then weassemble clean reads into unigene using mapping-first approaches methods.5. Alignment between unigenes and protein databases was performed, such as NCBInon-redundant protein (Nr) database, Uniprot protein database,the gene ontology(GO)database, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database.The sequence direction of unigenes, and expression and functional annotation of unigenes were then decided by the best alignment results.Results1. OD260/OD280total RNA is1.81, conc is42.75μg/ml, this result meetwithsequencing requirements.2. A total of132,007,609clean reads and91342unigenes weregenerated using Hiseq2000sequencing. The average length of these reads was101bp,proportion of available reads was76.1%, proportion of mapping reads was92.8%. Theseaverage length of these unigene was419bp, the GC content was43.78%.3. Based on asequence similarity search with known proteins, unigenes were identified after quality controlprocedures via the alignment of two databases. Overall,11866unigenes were mapped tonon-redundant protein (Nr) database.11294unigenes were mapped to Uniprot proteindatabase.6664unigenes were mapped to227Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways. Further biological process categories of GO were prominently representedin cellular process, metabolic process and multicellular organismal process. The KEGGpathways embodied metabolism (109members), genetic information processing (18members), environmental information processing (19members), cellular processes (12members), organismal systems (33members), and human diseases (36members).Conclusion1. This is the first study of the characteristic transcriptome of Echinococcusgranulosus protoscolex using Illumina sequencing technology.2. We obtained extensivetransciptome data from sequencing of Echinococcus granulosus protoscolex.3. Theseinformation will helpful to study the host-parasite of Echinococcus granulosus protoscolex,find novel diagnosis of hydatid disease. It also can provide novel apporaches for potentialdrug targets and vaccination against Echinococcus granulosus protoscolex.