Phylogenomic Analysis Of Balantidium Ctenopharyngodoni Based On Single-cell Transcriptome Sequencing

Balantidium ctenopharyngodoniis an obligate parasitic ciliate,living in the hindgut of grass carp(Ctenopharyngodon idella),which could cause hemorrhagic disease of grass carp.There are many previous studies which focused on morphology and pathology,however,the taxonomic status of B.ctenopharyngodonibases on the morphology characteristics and single genes.Especially,it is a blank for phylogenomics research based on orthologous genes among related species.Thus,using single-cell transcriptome sequencing,we obtained the transcriptome of B.ctenopharyngodoni.And according to the transcriptome,we performed a phylogenomics research.The main results are summarized as follows:1.Developed a single-cell transcriptome sequencing protocol which is suitable for B.ctenopharyngodoni.1)Clean cells for several times to remove food residue and bacteria by 0.65 % NaCl,then,get germ-free medium by filter the intestinal contents.This step will prolong the survival time of cells for cells to further digesting the intracellular foreign matter.2)Single-cell transcriptome sequencing were performed by HiSeq-4000,and 150 bp reads were obtained(In this study,we obtained 5.8 Gb clean data containing 1.9M reads pair).This protocol also fit for species that living in intricacy environment and can't be cultured to large population.2.Using single-cell transcriptome sequencing,we obtained the first transcriptome of B.ctenopharyngodoni and then carried out the functional annotation.After reads quality control,96.88 % of reads were used for transcriptome assembling to produce a 43.3 Mb raw transcriptome and then the transcriptome was optimized to get a 19.4 Mb transcriptome containing 38,426 transcripts,which was used for functional annotation.According to Gene Ontology(GO)and KEGG annotation,we predicted the metabolic pathways of B.ctenopharyngodoni,such as oxidative phosphorylation pathway,which is contributing to further functional analyses.3.Phylogenomics analysis about B.ctenopharyngodoni based on transcriptome.We identified 125 orthologous genes(48,102 aa in total)for phylogenomics analysis from B.ctenopharyngodoni transcriptome and other 26 ciliates genome.This phylogenomics analysis result shows that the relationship among Colpodea,Oligohymenophorea,Litostomatea,Spirotrichea,Heterotrichea and Protocruzia,and B.ctenopharyngodoni is close relationship toLitonotus sp.