Computational protocol: Comparative Transcriptome Analysis Reveals the Influence of Abscisic Acid on the Metabolism of Pigments, Ascorbic Acid and Folic Acid during Strawberry Fruit Ripening

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Protocol publication

[…] Low quality reads, including adaptors, low quality sequences (reads with more than 5% ambiguous bases) and reads with a Phred quality score Q ≤ 20 using the NGS QC toolkit [], were removed from the raw reads. Unigenes were obtained by assembling the resulting clean reads with the Trinity program [] and optimizing by TGICL []. Unigenes were aligned using BLASTx (E-value < 1e-5) to the NCBI non-redundant (nr) protein database. All of the unigenes were also compared with the protein databases Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), euKaryotic Ortholog Groups (KOG) and Pfam using the BLASTx algorithm with an E-value cut-off of 1e-5. Unigene expressions were normalized with the read counts per kilobase of exon model per million reads (RPKM) method []. Differentially expressed genes were analyzed among groups and the plots were performed according to the method described by Yu et al. []. The expressions of samples at three time points in each treatment or the expressions of samples with different treatments at the same time point (see the schematic diagram of data comparison in ) were compared by Chi-square analysis, only genes with p < 0.05 were identified as significantly differentially expressed. Deep sequencing data were further validated by quantitative reverse transcription PCR (qRT-PCR), and the primers used are listed in . The comparison between the sequencing data and qRT-PCR results are shown in . […]

Pipeline specifications

Software tools NGS QC Toolkit, Trinity, TGICL, BLASTX
Databases Pfam UniProt KEGG
Application Transcription analysis
Chemicals Abscisic Acid, Anthocyanins, Ascorbic Acid, Aspirin, Folic Acid, Masoprocol