| Neospora caninum is an intracellular parasitic which belongs to the protozoa apicomplexa phylum and distributed worldwide. Apart from leading to abortion, stillbirth, mummy foetus, weak tires and other serious reproductive disorders in different animals such as cattle, sheep, dogs, horses, foxs, white-tailed deer and some other animals, N. caninun can also leads to newborn pups motor nervous system disorders. N. caninum cause significant economic losses in animal husbandry, particularly to livestock.mi RNAs are one of the most important member of small non-coding RNAs, generally 21-25 nucleotides in length. They can regulate gene expression and guide various biological processes by programming the RNA induced silencing complex(RISC). Some of the mi RNA expression has been shown to be clear tissue-specific expression patterns as well as temporally regulated during expore mi RNA potential function in many species. However, there still have none studies on mi RNAs of N. caninum so far. Study on N. caninum mi RNAs would provide effective tool to evaluate the functions of related proteins in the parasite.In this study, we identified and analyzed the expression of mi RNAs in N. caninum tachyzoites, by using high through-put RNA sequencing and bioinformatic techniques. To survey mi RNAs in N. caninum, we constructed two small RNA libraries and sequenced using the solexa sequencing technology. A total of 55180517 s RNAs were obtained in the two small RNA libraries, among of them, 53160938 s RNAs were common in the two libraries, accounting for 96.34% of the total. A total of 28575212 and 34778655 high-quality sequence reads were obtained from two libraries, respectively. Among of them, there have 25678775(89.86%) and 29501742(84.43%) were clean sequence reads, 1125933(4.38%) and 954987(3.24%) were unique reads, respectively.Clean reads were obtained and mapped to the reference genome, as a result, 5889027(22.93%) and 13412613(45.46%) can mapped to the reference genome successfully. Among of them, 4622636(78.50%) and 10510023(78.36%) reads were unique mapped, 1266391(21.50%) and 2902590(21.64%) reads were multiple mapped, respectively. Based on their genomic locations, these reads were categorized into four types named intergenic, intronic, CDS, and noncoding-exon, and most of reads were located in intergenic regions.The result of mapping genome of N. caninum showed that most reads located at intergenic regions, widely distributed in 14 chromosomes, including the most distribution on chromosome Ia and the least on chromosome IV. The length of small RNAs mainly varied from 18 to 30 nt in the two libraries. A total of 10 conserved mi RNAs in 10 metazoan mi RNA families and 290 novel mi RNAs were identified in N. caninim by bioinformatics analysis. Among of them, we found most of mi RNAs were derived from just one hairpin arm, 66 were derived from both arms. We use real-time PCR method based on SYBR Green fluorescent dye to confirm the Solexa sequencing results, the results are consistent with high through-put sequencing analysis results, showed that the sequencing data is reliable.In summary, this study presents a first systematic analysis of mi RNAs in N. caninum by combination of Solexa high through-put sequencing and bioinformatics analysis, providing insight information of the parasite biology. The data of this study have paved a new way for future research in N. caninum, and hopefully facilitate the discovery of potential anti-parasitic drug targets. |
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