Computational protocol: De novo transcriptome analysis of Perna viridis highlights tissue-specific patterns for environmental studies

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Protocol publication

[…] The raw reads from the six samples were merged, and then each read was pre-processed by the trimming of adaptors and the removal of 10 bp from the 3’ end. Low-quality reads were filtered out based on three criteria: reads with more than 5% unknown “N” bases; reads having more than 50% bases with a quality value less than 10; and reads < 35 bp. The resulting high-quality reads were then used for de novo assembly with Trinity version trinityrnaseq_r2012-10-25 [] using default parameters. Trinity used the greedy k-mer extension (k-mer 25) to assemble raw reads into unique sequences of transcripts, followed by clustering of the contigs to construct complete de Bruijn graphs, and finally processed the individual graphs to generate full-length transcripts. The assembled transcripts were further clustered using CD-HIT [] to reduce redundancy. Briefly, CD-HIT employed a 'longest sequence first' algorithm by identifying a representative sequence and then clustered each subsequent sequence as a redundant or a new cluster sequence based on a similarity score set at ≥ 95% for our analysis. The longest transcript in each gene cluster defined by Trinity was selected as a “unigene” for downstream analysis.The annotation of assembled transcripts/unigenes was performed based on sequence similarity searches using BLASTx against the NCBI nr database (08 February 2013), and BLASTn and tBLASTn searches against the EST databases from the molluscan genome projects of Aplysia californica (GenBank Accession Number AASC00000000, 255,605 EST entries), Crassostrea gigas (AFTI00000000, 206,647 EST entries) and Lottia gigantea (AMQO00000000, 252,091 EST entries), with an E-value threshold of 1 × 10−5 and a high-scoring segment pairs cut-off length of 33. Functional classification was carried out based on GO using Blast2GO []. GO terms were assigned and annotated to the transcripts/unigenes according to three main categories: cell component; molecular function; and biological process. The presence of a conserved domain in transcript/unigene was analysed using InterProScan [] within Blast2GO. KEGG pathways were assigned to the transcripts using the online KEGG Automatic Annotation Sever (KAAS; http://www.genome.jp/kegg/kaas/) using the bi-directional best-hit method. [...] The high-quality reads from the six libraries (2 sexes × 3 tissues) were individually mapped to the global assembled de novo trancriptome to estimate the abundance of transcripts/unigenes using RNA-Seq by Expectation Maximization (RSEM) version 1.2.3 [] with a pipeline script provided by Trinity. High-quality reads were mapped to the transcripts and the resulting alignment file was inputted into RSEM to calculate the expression value for each transcript. Expression level was presented in a normalized expressed value as FPKM, which corrects for read differences in libraries and gene length. A transcript was only considered expressed when the FPKM cut-off value was ≥ 0.05. [...] Tissue-specific expression patterns for 15 selected genes (Additional file ) associated with stress responses/detoxification were revealed using qPCR analysis. Mussel samples were chosen from selected treatments (i.e., control, 730 μg/L CdCl2, 13 mg/L nZnO, 40 μg/L DDT, and 0.32 μg/L TPTCl) of the exposed-type that used in the library generation. Primer pairs for qPCR assays were designed using Primer3 version 0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/). For amplification, 1 mM reverse and forward primers and 2 μL of cDNA were used per 20 μL reaction on a CFX96™ Real-Time System (Bio-Rad, Hercules, USA) with iQ™ SYBR® green supermix (Bio-Rad, Philadelphia, USA), following the manufacturer’s recommended protocol and default settings . Thermal cycling conditions involved 3 min of pre-heating at 95°C, followed by 40 cycles of amplification (10 s at 95°C, 30 s at 60°C); a melt curve analysis was performed after the 40th cycle (10 s at 95°C, then 65–95°C in 0.5°C increments every 5 s). Relative expression levels of target genes were calculated using the 2-ΔΔCT method [], with the 18S rRNA gene used as a reference and pooled samples from all tissues as calibrator samples. The 18S gene was shown to be a suitable reference gene in mussels and its expression was stable among different chemical treatments [, ]. Expression of the 18S gene was not significantly different among the various control and treatment groups (ANOVA, p > 0.05), and between sexes (t-test, p > 0.05). […]

Pipeline specifications

Software tools Trinity, CD-HIT, BLASTX, BLASTN, TBLASTN, Blast2GO, InterProScan, KAAS, RSEM
Databases KEGG
Applications RNA-seq analysis, Amino acid sequence alignment
Organisms Perna viridis
Diseases Drug-Related Side Effects and Adverse Reactions