De Novo Assembly of the Sea Cucumber Apostichopus japonicus Hemocytes Transcriptome to Identify miRNA Targets Associated with Skin Ulceration Syndrome

Pengjuan Zhang,Chenghua Li,Lin Zhu,Xiurong Su,Taiwu Li,Ye Li,Chunhua Jin,Christian Schönbach

doi:10.1371/journal.pone.0073506

Pengjuan Zhang, Chenghua Li + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0073506

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Abstract

Background De novo transcriptome sequencing is a robust method of predicting miRNA target genes, especially samples without reference genomes. Differentially expressed miRNAs have been previously identified in hemocytes collected from healthy skin and from skin affected by skin ulceration syndrome (SUS) in Apostichopus japonicus . Target identification for these differentially expressed miRNAs is a major challenge for this non-model organism.Methodology/Principal FindingsTo thoroughly understand the function of miRNAs, a normalized cDNA library was sequenced with the Illumina Hiseq2000 technology. A total of 91,098,474 clean reads corresponding to 251,148 unigenes, each with an average length of 494bp, were obtained. Blastx analysis against a nonredundant (nr) NCBI protein database revealed that in this set, 52,680 unigenes coded for 3,893 annotated proteins. Two digital gene expression (DGE) libraries from healthy and SUS samples showed that 4,858 of the unigenes were expressed at significantly different levels; 2,163 were significantly up-regulated, while 2,695 were significantly down-regulated. The computational prediction of miRNA targets from these differentially expressed genes identified 732 unigenes as the targets of 57 conserved and 8 putative novel miRNA families, including spu-miRNA-31 and spu-miRNA-2008.ConclusionThis study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The DGE assembly data represent a substantial increase in the genomic resources available for this species and will provide insights into the gene expression profile analysis and the miRNAs function annotations of further studies.

Highlights

RNA-seq is a powerful and rapidly developing approach for unbiased transcriptome analysis
The identification and characterization of candidate genes or miRNAs involved in skin ulceration syndrome (SUS) outbreaks would represent the first step in understanding the genetic basis of this process in sea cucumbers
After trimming the adapter sequences and removing low-quality sequences, 91,098,474 clean reads remained for the normalized cDNA library, with average GC content of 38.91%

Summary

Introduction

RNA-seq is a powerful and rapidly developing approach for unbiased transcriptome analysis. A normalized cDNA library from the same sample used for our miRNA analysis [14] was constructed and sequenced with Illumina Hiseq2000. The sequence reads were assembled and annotated by a BLAST analysis against the NCBI NR database; third, two digital gene expression (DGE) libraries were sequenced to screen differentially expressed genes and to predict miRNA targets.

Results

Conclusion