Computational protocol: Transcriptome profiling of sulfate deprivation responses in two agarophytes Gracilaria changii and Gracilaria salicornia (Rhodophyta)

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Protocol publication

[…] Massive paired-end RNA Seq was performed on each Gracilaria sample using Illumina HiSeq 2000 platform (Illumina, San Diego, CA, USA) at a read length of 90 bp. The RNA-Seq data were deposited in European Nucleotide Archive (ENA) under the accession number PRJEB13899. The raw reads obtained from RNA-Seq were analysed with FastX toolkit v0.0.13.2 (http://hannonlab.cshl.edu/fastx_toolkit/) to remove low-quality reads with Phred quality scores <20, adaptor and ambiguous bases ‘N’. De novo assembly of clean reads obtained from the four samples was performed with Velvet (v1.2.08) with default parameters and optimized k-mers at 67, 69, 65, and 65 for CC, CT, SC, and ST, respectively. Assembled sequences that were less than 100 nucleotides were discarded. Iterative clustering of contigs from control and treated samples was performed with The Gene Index Clustering Tool (TGICL) to generate two sets of non-redundant contigs (unigenes) for G. changii and G. salicornia, respectively.Sequence based alignments were performed against the following public databases: non-redundant protein sequences (NR), UniProtKB/SwissProt (Swiss-Prot), GO and KEGG. BLASTx program with default settings and a cut-off E-value of 10−5 was used to match the nucleotide sequences to those in the NR and SwissProt databases at the NCBI. The GO annotation was performed with BLAST2GO based on the most significant SwissProt matches and the results were visualized using Web Gene Ontology Annotation Plot (WEGO). KEGG annotation was performed with the KEGG Automatic Annotation Server (KAAS, http://www.genome.jp/kegg/kaas/). The most significant and informative matches were used to assign putative function to each unigene. Reciprocal BLASTn was conducted on the two sets of unigene with a cut-off E-value of 10−30. [...] The paired-end RNA-Seq reads from each sample were mapped to respective transcriptomes with Bowtie2. The number of reads mapped to each unigene was converted into RPKM, to normalise the differences in transcript length and sequencing depth. DEGs between control and sulfate-deprivated samples of G. changii and G. salicornia, respectively, were identified by DEGseq, an R package which uses the MA-plot-based method with Random Sampling model (MARS). In the absence of replicates, DEGseq treated the counts of reads that were mapped to a gene from two samples (control and sulfate-deprivated samples in this study) as independent values and tested them with the following hypotheses, H0: p1 = p2 = p versus H1: p1≠p2; p1 and p2 denote the probability that a read obtained from sample 1 (e.g. control) and 2 (e.g. sulfate-deprivated), respectively. The two estimates generated from above were then used for the calculation of Z-score which can be converted to two-sided p-value. Transcripts with RPKM read ≥15 in at least one of the samples, p-value less than 0.001 and fold change ≥1.5-fold were defined as DEGs (). [...] Verification of DEGs was performed on the same RNA samples for RNA-Seq experiment. Affinity Script QPCR cDNA Synthesis Kit (Agilent Technologies, USA) was used to reverse transcribe 2.5 μg of total RNA from each sample into the first strand cDNA. The DEGs and the primers used for qRT-PCR analysis are listed in . Primer3 software ver. 0.4.0 (http://frodo.wi.mit.edu) was used to design primers that are specific to the 3′-untranslated region flanking the open reading frame, to avoid amplification from possible isoforms/gene family members. Melting curve and standard curve (with serial dilutions of cDNA samples) were generated to check for amplification of single PCR product, absence of primer dimers and PCR efficiency (90–105%).All qRT-PCR analyses were performed using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, USA) and iQTM5 real time detection system (Bio-Rad, USA). The reaction mixture (20 μl) consisted of 100 ng of first-strand cDNA template, 1X Brilliant III Ultra-Fast SYBR Green QPCR master mix, 200–400 nM of each respective primers and molecular grade H2O (bioWORLD, USA). The PCR amplification was performed with the following conditions: initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 5 sec and 60 °C for 10 sec; and a melt curve analysis with temperature increasing from 55 °C to 95 °C with an increment of 0.5 °C/10 sec. A control sample without template was included in all experiments to rule out the presence of contaminants and primer-dimer.Two internal reference genes for G. changii (encoding histone and cytosolic fructose-1,6-biphosphatase, respectively) and G. salicornia (encoding 40S ribosomal protein S15a and tRNA-dihydrouridine synthase-1-like, respectively) were selected based on their stability ranking and pairwise variation as analysed by geNorm version 3.5. All samples in the qRT-PCR analysis were performed in three technical replicates. The raw Ct values obtained were analysed using 2−ΔΔCt method, and normalized against the expression of internal reference genes. The normalized expression level in all samples was expressed as relative fold change to that in the control samples. […]

Pipeline specifications

Software tools FASTX-Toolkit, Velvet, TGICL, BLASTX, Blast2GO, WEGO, KAAS, BLASTN, Bowtie2, DEGseq, Primer3
Databases ENA UniProt UniProtKB KEGG
Applications RNA-seq analysis, qPCR, Transcription analysis
Organisms Gracilaria salicornia
Chemicals Amino Acids, Carbon, Sulfur