Constructing and detecting a cDNA library for mites

Li, Hu; Zhao, Yongtao; Juan, Carlos; Yang, Yaolong; Li, Chen; Zhang, Lu

doi:10.1007/s00436-015-4621-x

Cited by 9 publications

(2 citation statements)

References 15 publications

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“…Li et al sequenced 33 P. ovis var. cuniculi ESTs by constructing a cDNA library [46]. Thus, overall, relatively few P. ovis sequences were obtained in previous investigations.…”

Section: Discussionmentioning

confidence: 99%

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Preliminary analysis of Psoroptes ovis transcriptome in different developmental stages

et al. 2016

Parasites Vectors

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BackgroundPsoroptic mange is a chronic, refractory, contagious and infectious disease mainly caused by the mange mite Psoroptes ovis, which can infect horses, sheep, buffaloes, rabbits, other domestic animals, deer, wild camels, foxes, minks, lemurs, alpacas, elks and other wild animals. Features of the disease include intense pruritus and dermatitis, depilation and hyperkeratosis, which ultimately result in emaciation or death caused by secondary bacterial infections. The infestation is usually transmitted by close contact between animals. Psoroptic mange is widespread in the world. In this paper, the transcriptome of P. ovis is described following sequencing and analysis of transcripts from samples of larvae (i.e. the Pso_L group) and nymphs and adults (i.e. the Pso_N_A group). The study describes differentially expressed genes (DEGs) and genes encoding allergens, which help understanding the biology of P. ovis and lay foundations for the development of vaccine antigens and drug target screening.MethodsThe transcriptome of P. ovis was assembled and analyzed using bioinformatic tools. The unigenes of P. ovis from each developmental stage and the unigenes differentially between developmental stages were compared with allergen protein sequences contained in the allergen database website to predict potential allergens.ResultsWe identified 38,836 unigenes, whose mean length was 825 bp. On the basis of sequence similarity with seven databases, a total of 17,366 unigenes were annotated. A total of 1,316 DEGs were identified, including 496 upregulated and 820 downregulated in the Pso_L group compared with the Pso_N_A group. We predicted 205 allergens genes in the two developmental stages similar to genes from other mites and ticks, of these, 14 were among the upregulated DEGs and 26 among the downregulated DEGs.ConclusionThis study provides a reference transcriptome of P. ovis in absence of a reference genome. The analysis of DEGs and putative allergen genes may lay the foundation for studies of functional genomics, immunity and gene expression profiles of this parasitic mite species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1856-z) contains supplementary material, which is available to authorized users.

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“…Li et al sequenced 33 P. ovis var. cuniculi ESTs by constructing a cDNA library [46]. Thus, overall, relatively few P. ovis sequences were obtained in previous investigations.…”

Section: Discussionmentioning

confidence: 99%

“…cDNA libraries of P. ovis were constructed by Kenyon et al [45] and Burgess et al [20], and a cDNA library of P. ovis var. cuniculi was constructed by Hu et al[46] . The experimental materials used in these studies were all mixed life-cycle stages of P. ovis , including larvae, nymphs and adults.…”

Section: Discussionmentioning

confidence: 99%

Preliminary analysis of Psoroptes ovis transcriptome in different developmental stages

et al. 2016

Parasites Vectors

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Improvement on the extraction method of RNA in mites and its quality test

Zhao

Yang

et al. 2015

Parasitol Res

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To solve the long-existing difficult problems in extracting RNA and constructing a complementary DNA (cDNA) library for trace mites, we conducted a further comparative experiment among three RNA extraction methods (TRIzol method, Omega method, and Azanno method) based on our previous attempts at the construction of cDNA library of mites, with Psoroptes cuniculi still used as the experimental subject. By subsequently decreasing the number of mites, the least number of mites needed for RNA extraction of each method were found by criteria of completeness, concentration, and purity of the extracted RNA. Specific primers were designed according to the allergen Pso c1, Pso c2, and Actin gene sequences of Psoroptes to test the reliability of cDNA library. The results showed that Azanno method needed only 10 mites with sensitivity 204 times higher than previously used TRIzol method and 20 times higher than Omega method; clear RNA band was detected by agarose gel electrophoresis; and ultraviolet spectrophotometer determination showed that RNA concentration, 260/280, and 260/230 were in the range of 102 to 166 ng/μl, 1.83 to 1.99, and 1.49 to 1.72, respectively. Finally, specific primers detection showed that the amplified sequences had 98.33, 98.19, and 99.52% identities with those of P. cuniculi or Psoroptes ovis in GenBank, respectively, indicating that the cDNA library constructed using 10 mites was successful and it could meet the requirements for molecular biology research. Therefore, we concluded that Azanno method was more effective than TRIzol method and Omega method in RNA extraction and cDNA library construction of trace mites.

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De novo RNA-Seq and functional annotation of Sarcoptes scabiei canis

Zhao

Yang

et al. 2016

Parasitol Res

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The transcriptomic data of Sarcoptes is still lacking in the public database due to the difficulty in extracting high-quality RNA from tiny mites with thick chitin. In this study, total RNA was extracted from live Sarcoptes mites for quality assessment, RNA-Seq, functional annotation, and coding region (CD) prediction and verification. The results showed that the sample JMQ-lngm was qualified for cDNA library construction. Firstly, Agilent 2100 detection showed that the RNA baseline was smooth and the 18S peak was single. Second, the Illumina platform generated 65.78M clean reads and 20,826 unigenes with 35.43M were assembled, occupying 62.98 % of the 56.26M genome. In total, 15,034 unigenes were annotated in seven functional databases. Finally, 13,122 CDs were detected in the 20,826 unigenes, of which 70 complete CDs were matched with Sarcoptes manually in non-redundant nucleotide (NT). Three CDs with indels ≥10 bp were verified. Those results indicated that peritrophin sequences of JMQ-lngm missed 35 bp during the assembly; the pressure-sensitive sodium channel sequences of all the six Sarcoptes scabiei canis isolates were confirmed to be 90 bp shorter than that of a Sarcoptes scabiei hominis isolate; three introns remained in PH chlorine ion channel gating sequences of JMQ-lngm. Moreover, the allergen gene prediction for JMQ-lngm indicated that 61 unigenes were matched with 19 allergen genes of Dermatophagoides, of which Der 1, Der 3, Der 8, and Der 10 had been confirmed in NT. In conclusion, this study successfully completed the RNA-Seq and functional annotation of S. s. canis for the first time, which provides molecular data for future studies on the identification and pathogenic genes of Sarcoptidae.

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Constructing and detecting a cDNA library for mites

Cited by 9 publications

References 15 publications

Preliminary analysis of Psoroptes ovis transcriptome in different developmental stages

Preliminary analysis of Psoroptes ovis transcriptome in different developmental stages

Improvement on the extraction method of RNA in mites and its quality test

De novo RNA-Seq and functional annotation of Sarcoptes scabiei canis

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