De novo assembly of Manila clam (<i>Ruditapes philippinarum</i>) and protozoan parasite (<i>Perkinsus olseni</i>) transcriptome from RNA-Seq data

• Main Authors: Abul Farah Md. Hasanuzzaman, Peter Harrison, Antonio Gómez Tato, Jose Antonio Alvarez Dios, Asunción Cao
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2014-06-01
• Series: Frontiers in Marine Science
• Subjects: RNA-Seq; host-parasite interaction; Ruditapes philippinarum; de novo transcriptome; oligo-microarray; Perkinsus olseni; unigenes annotation
• Online Access: http://journal.frontiersin.org/Journal/10.3389/conf.fmars.2014.02.00109/full

Manila clam (<i>Ruditapes philippinarum</i>) with its high commercial value has become a major contributor to the world´s clam production from both bivalve fishery and aquaculture ventures. But, the production of manila clam is at greater risk from diseases, especially caused by <i>Perkinsus olseni</i>. Considering the limited genomic resources of <i>R. philippinarum</i> and <i>P. olseni</i> as well as the increasing interest in immune-genes response in the host, the present work was conducted to analyze the transcriptome profile of <i>R. philippinarum</i> and of <i>P. olseni</i> using RNA-Seq technology. Data obtained will be used to construct transcriptome databases and to design oligo-microarrays both in the host and the parasite for understanding their transcriptional expressions through host-pathogen interactions. cDNA libraries were synthesized using RNA aliquots of clam haemocytes and perkinsus trophozoites collected from different experimental conditions (<i>in vitro, in vivo</i> and natural environment) for further sequencing carried out on Illumina HiSeq 2000 (Oxford Genomics Center, UK). A total of 131,037,742 pair-end reads for <i>R. philippinarum</i> and 67,840,472 for <i>P. olseni</i> were produced. De novo assembly was evaluated using different parameters of Trinity and Abyss assembler programs. De novo transcriptome of clam was further undergone through filtering steps by expression (> 5 FPKM), and length (500 bp) for improving transcriptome quality. CAP3 software was executed to cluster final de novo transcriptomes. Trinity+CAP3 assembly was the best approach after manual evaluation of 30 annotated contigs randomly sampled from each strategy and was chosen finally to assemble transcriptomes. 33,182 unique transcripts (3,031 contigs and 30,151 singletons) from de novo clam transcriptome, and 47,590 unique transcripts (7,251 contigs and 40,339 singletons) from perkinsus transcriptome were obtained. Among them; 3,031 (9.13%) unigenes of <i>R. philippinarum</i>, and 7,251 (15.24%) of <i>P. olseni</i> were annotated with NCBI nucleotide (nt) databases based on e-value <10-5. This is the first comprehensive transcriptome reported for <i>P. olseni</i>, and also the first one from RNA-Seq for <i>R. philippinarum</i>. Using these de novo transcriptome resources, microarrays will be designed for all the unique sequences and their splice variants identified in each database. The comparative analysis will provide new insights into understanding molecular mechanisms of gene expressions through clam-perkinsus interactions.